Methods for enhancing the production and consumer traits of plants

ABSTRACT

The invention provides methods for producing plants, plant materials and seeds that have multiple desirable attributes for consumers of these products, as well as for commercial plant growers, and to improved plants, plant materials and seeds that are produced by these methods. These inventive methods provide hybrid plants, plant materials and seeds having the mutant shrunken-2i (sh2-i) allele incorporated into their genomes, preferably sequentially along with one or more other mutant alleles, such as the sugary-1 (su1), sugary enhancer-1 (se1) and/or shrunken-2 (sh2) alleles, and that have multiple beneficial traits, including an extended sugar retention ability at the post prime eating stage and a significantly enhanced vigor and fitness to the plant, plant material and/or seed during seed germination, seedling emergence from soil, and plant development.

REFERENCE TO SEQUENCE LISTING SUBMITTED ON A COMPACT DISC

All material that is present in a replacement Sequence Listing in acomputer readable form (CRF), in a form of two identical CD-R compactdiscs, that was filed with the U.S. Patent and Trademark Office (“PatentOffice”) on Jul. 14, 2010 for related parent U.S. patent applicationSer. No. 12/462,959, filed on Aug. 12, 2009, which was created usingPatentIn Version 3.5 computer software, and which has been determined tobe compliant by the Patent Office, is hereby incorporated into thisapplication by reference in its entirety. Each of these two identicalCD-R compact discs is separately identified below.

Copy 1 Replacement Jul. 14, 2010

-   -   Patent Applicant: Bryant Jerome Long    -   Patent Application: U.S. Ser. No. 12/462,959    -   Filed: Aug. 12, 2009    -   Title: Methods for Enhancing the Production and Consumer Traits        of Plants    -   Data Recordation Date: Jul. 14, 2010    -   Machine Format Used: IBM-PC    -   Operating System Used: Microsoft Windows XP    -   File Name: 3001401-00003—CRF Sequence Listing . . .    -   File Size: 35.7 KB    -   Number of Sequences: 29

Copy 2 Replacement Jul. 14, 2010

-   -   Patent Applicant: Bryant Jerome Long    -   Patent Application: U.S. Ser. No. 12/462,959    -   Filed: Aug. 12, 2009    -   Title: Methods for Enhancing the Production and Consumer Traits        of Plants    -   Data Recordation Date: Jul. 14, 2010    -   Machine Format Used: IBM-PC    -   Operating System Used: Microsoft Windows XP    -   File Name: 3001401-00003—CRF Sequence Listing . . .    -   File Size: 35.7 KB    -   Number of Sequences: 29—

BACKGROUND OF THE INVENTION

1. Field of the Invention

This application is a continuation-in-part patent application of pendingnon-provisional patent application U.S. Ser. No. 12/462,959, filed onAug. 12, 2009. This continuation-in-part patent application claims thebenefit of prior non-provisional patent application U.S. Ser. No.12/462,959, filed on Aug. 12, 2009, which patent application is herebyincorporated into this continuation-in-part patent application in itsentirety by reference herein, including all drawings, all seed and/orother deposits made with the American Type Culture Collection (ATCC) andall sequence listings.

The present invention is directed to unique methods for producingplants, plant materials and seeds that very advantageously have enhancedproduction and consumer traits, such as an improved vigor and hardinessof the seeds, an enhanced seedling emergence and plant growth in bothwarm and cold soils (i.e., in different types of climates and atdifferent temperatures), combined with a superior taste quality andkernel pericarp tenderness, and to improved plants, plant materials andseeds that are produced in accordance with these methods, or that havesuch traits. The invention provides a novel approach for combining aunique assembly of genetic traits in plants, plant materials and seeds,using double, triple and other allelic combinations, resulting inenhanced growing potential along with desirable consumer qualities.

2. Background

Maize

Corn (maize) is one of the most diverse grain crops that is present innature, and there are a number of different types of corn, which aregenerally classified by characteristics of their kernel endosperm. Themost common types of corn include flint, flour, dent, pop, sweet, waxyand pod. The physical appearance of each kernel type is determined byits endosperm pattern, quality and quantity.

Sweet corn is a kind of corn plant that is classified as Zea mays, var.rugosa, and that has white, yellow or bi-colored kernels that are sweetwhen they are in the immature milky stage as a result of having a highsugar content. Higher levels of sugar in the sweet corn kernels resultin a lower osmotic potential, causing greater water uptake into thekernels. Sweet corn is typically eaten by human beings as a vegetable,either directly from the maize cob, or by having the sweet kernelsremoved from the cob, and is a major vegetable crop that is grown allover the world primarily for fresh consumption, rather than as animalfeed or for flour production.

Sweet corn occurs as a spontaneous mutation in field corn, and can bethe result of naturally-occurring mutations in the genes that controlconversion of sugar to starch inside the endosperm of the corn kernel.Unlike field corn varieties, which are intended for livestock, and aretypically harvested when their kernels are dry and fully mature (at thedent stage), sweet corn is typically picked when it is immature (at themilk stage), and eaten as a vegetable, rather than as a grain. Becausethe process of maturation involves converting sugar into starch, sweetcorn typically stores poorly, and must be eaten in a fresh, canned orfrozen manner before the kernels become tough and/or starchy. Followingharvest, or if left on the stalk too long, sucrose in standard sweetcorn becomes rapidly converted to starch. Kernels can lose as much as50% of their sucrose at room temperature at around 24 hours afterharvest.

Open pollinated (non-hybrid) varieties of white sweet corn started tobecome widely available in the United States in the 19th century. Two ofthe most enduring varieties, which are still available today, areCountry Gentleman (a Shoepeg corn with small, white kernels in irregularrows) and Stowell's Evergreen. Sweet corn production in the 20th centurywas influenced by the following key developments:

-   -   (i) hybridization, which allowed for more uniform maturity,        improved quality and disease resistance; and    -   (ii) identification of the separate gene mutations that are        responsible for sweetness in corn, and the ability to breed        varieties based on these characteristics, for example:        -   su (normal sugary);        -   se (sugary enhanced); and        -   sh2 (shrunken-2).            There are currently hundreds of varieties of sweet corn,            with more varieties continuously being developed.

The fruit of the sweet corn plant is the corn kernel, and the earconsists of a collection of kernels on the cob. Because corn is amonocot, there is always an even number of rows of kernels. The ear iscovered by tightly wrapped leaves (the husk).

Sweet corn has significant antioxidant activity, which can reduce thepossibilities of developing heart disease and/or cancer. It alsoreleases increased levels of ferulic acid, which provides additionalhealth benefits.

There are several known genetic mutations that are responsible for thevarious types of sweet corn. Early varieties were the result of themutant su1 (sugary-1) allele. Conventional su1 varieties contain about5-10% sugar by weight.

Varieties of sweet corn that contain the shrunken-2 (sh2) gene typicallyproduce higher than normal levels of sugar, and have a longer shelflife, in comparison with conventional sweet corn, and are frequentlyreferred to as supersweet varieties. One specific gene in sweet corn,the shrunken-2 (sh2) gene, causes the mature corn kernel to dry andshrivel as it matures past the milky stage, which is an undesirabletrait for seedling germination, early emergence and plant growth. Theendosperm of conventional sh2 sweet corn kernels store less amounts ofstarch, and from about 4 to about 10 times more sugar, than conventionalsu1 sweet corn. This has permitted the long-distance shipping of sweetcorn, and has enabled manufacturers to can sweet corn without addingextra sugar or salt to it.

The third gene mutation is the se1 (sugary enhanced-1) allele, which isincorporated in the genome of Everlasting Heritage varieties.Conventional sweet corn varieties with the se1 alleles typically have alonger storage life, and contain from about 12% to about 20% sugar(i.e., a much higher sugar level in comparison with the conventional su1varieties).

All of the alleles that are responsible for sweet corn are recessive, sosweet corn must generally be isolated from any field corn varieties thatrelease pollen at the same time. The endosperm develops from genes fromboth parents (male and female), and kernels will generally be tough andstarchy.

Maize was first classified according to the carbohydrate that is storedin its endosperm. The most distinguishable sugar component that ispresent in sweet corn is sucrose, which accounts for the vast majorityof its sweetness differentiation. (Abbott and Cobb, Inc., PlantProtection No. 9600094 (1998).) The reducing free sugars, glucose andfructose, are present in sweet corn in significantly lower levels. Thesereducing sugars primarily result from the natural hydrolysis of sucrose.

Most commercially-available sweet corns are based upon single recessivealleles that alter carbohydrate composition of the endosperm. Untilabout thirty years ago, sweet corn was typically defined by the su1mutant allele located on chromosome four. Although the primarydifference between sweet corn and field corn is historically due to asingle gene, there are many other genes, both exhibiting major and minoreffects that differentiate the two classifications of corn. These genesaffect the overall eating quality of the corn (flavor, texture,tenderness and the like) along with the appearance of the kernel, earand plant, as well as the viability of the seed. Sweet corn isdifferentiated mainly from other types of corn, i.e., flint, flour,dent, waxy, pop, and the like, by the presence of genes that typicallyalter endosperm starch synthesis and its use as a vegetable for humanconsumption.

Many of the endosperm mutant genes in maize (and in other crop plants)that are presently being used commercially in different sweet cornvarieties, and affect endosperm carbohydrate synthesis, are listed inTable 1 below. These endosperm mutations all are believed to affectstarch synthesis during kernel development, and have been characterizedand mapped in maize.

TABLE 1 Sweet Corn Endosperm Mutant Genes Used Commercially Gene GeneSymbol Chromosome Amylose-extender ae 5 Brittle bt 5 Brittle-2 bt2 4Dull du 10 Shrunken-2 sh2 3 Sugary-1 su1 4 Sugary enhancer-1 se1 2 Waxywx 9Of the endosperm mutants that are listed in Table 1, the mutants thatare most widely used commercially are sugary-1 (su 1), sugary enhancer-1(se1) and shrunken-2 (sh2).

Endosperm Mutants

Sweet corn eating quality is typically largely determined by itssweetness which, in turn, is affected by the amount of sugar and starchthat are present in the endosperm of its kernels. The major and,therefore, most distinguishable sugar component in sweet corn issucrose, which accounts for most of its sweetness differentiation. Thereducing sugars, glucose and fructose, are typically present in sweetcorn in significantly lower levels in comparison with sucrose. Thesereducing sugars typically result from the natural hydrolysis of sucrose.Sugary-1 (su1), sugary enhancer-1 (se1), and shrunken-2 (sh2) mutantalleles have been determined to markedly increase sugar content, anddecrease starch levels, in sweet corn in comparison with corn that doesnot include one or more of these mutant alleles in its genome.

Another determining characteristic of sweet corn is that its kernels(seeds) tend to be markedly more tender in comparison with other typesof corn. Significant degrees of tenderness levels may occur within sweetcorn varieties expressing one or more of the above three (or other)endosperm mutant alleles. Elevated sugar levels and increased kernelpericarp tenderness that typically occur in these varieties generallyallows for extended holding abilities or shelf lives. An extendedholding ability or shelf life, both on and off of a corn plant (or otherproduction or distribution facility), is generally extremely importantwhen considering shipping sweet corn varieties over potentially longgeographical distances to market, for example, across the United States,or from one country to another country.

Currently, for reasons related to elevated sugar and pericarp tendernesslevels, the shrunken-2 (sh2) sweet corn class is the most popular typeof sweet corn commercially available. However, as is describedhereinbelow, such class has some very significant disadvantages, whichare overcome by the processes and products of the present invention.

Su1 (Sugary-1) Mutant Gene

Sweet corn in its original form differs from field corn primarily by thepresence of a single recessive mutant gene, sugary-1 (su1). Therecessive sugary-1 (su1) genotype that is present in sweet corn has aneffect of retarding (significantly slowing) a normal conversion of sugarinto starch during endosperm development, which very desirably resultsin a sweet taste, rather than in a starchy taste. This gene has beencloned and mapped to the short arm of chromosome 4 in sweet corn, andits genomic sequence and amino acid sequence translation are set forthin U.S. Pat. No. 5,912,413 entitled “Isolation of the SU1 StarchDebranching Enzyme, the Product of the Maize Gene Sugary 1,” and herein.

The sugary (su1) gene encodes a Class II starch debranching enzyme thatis active in cellular plastids. It is an isoamylase that hydrolyzes theα-(1,6) branch linkages in starch during starch synthesis. (J A Shultzet al., “Current Models for Starch Synthesis and the Sugary Enhancer 1(se1) Mutation in Zea mays,” Plant Physiology and Biochemistry 42(6),457-464 (2004).)

The sugary-1 (su1) gene confers a moderate increase in overall sugarlevels to corn kernels, but disadvantageously has only about one half ofthe total sugar content of “supersweet” corn varieties conferred by theShrunken-2 (sh2) gene, which is significantly less desirable to cornconsumers. Also disadvantageously, the conversion of sugar to starch inthe corn kernels is comparatively rapid, generally resulting in areduced or narrow harvest window before the sweetness (i.e., sugarlevels) of the corn kernels deteriorates after the prime eating stage(at approximately 75% kernel moisture).

The sugary-1 (su1) gene, contrary to its name, therefore, does notgenerally result in exceptionally high levels of sugars. However, itdoes generally result in greatly increased levels of phytoglycogen orwater soluble polysaccharides (WSP). (W. F. Tracey, In A. R. Hellauer(ED) Specialty Corns (CRC Press, Boca Raton, Fla.) 147-187 (1994).)Phytoglycogen and WSP tend to give the endosperm of the kernels ofconventional su1 sweet corn varieties the smooth texture and creaminesscharacteristic of traditional sweet corn varieties. Mature endosperm ofnon-mutant corn generally contains approximately 2% WSP, whereas cornlines that are homozygous for the sugary-1 (su1) gene may contain up toapproximately 35% WSP.

The mutant sugary-1 (su1) gene present in conventional varieties ofsweet corn disadvantageously tends to result in a significantly reducedseed quality and use compared to field corn varietal counterparts.

Additional information about the recessive sugary-1 (su1) gene ispresent in M. G. James et al., “Characterization of the Maize GeneSugary-1, a Determinant of Starch Composition in Kernels,” The PlantCell, Vol. 7, 417-429 (1995); and D. Pan et al., “A Debranching EnzymeDeficiency in Endosperms of the Sugary-1 Mutants of Maize,” PlantPhysiol. 74(2), 324-328 (1984).

Se1 (Sugary Enhancer-1) Mutant Gene

The sugary enhancer-1 (se1) mutant gene is a recessive modifier of thesugary-1 (su1) gene mutation. (J A Shultz et al., “Current Models forStarch Synthesis and the Sugary Enhancer 1 (se1) Mutation in Zea mays,”supra.). The effects of the sugary enhancer-1 (se1) mutant gene wereoriginally observed in 111677a, a line derived from a three-way cross(Bolivia 1035X Illinois 44b X Illinois 442a). (J. W. Gonzales et al., “ANew Inbred Line With High Sugar Content in Sweet Corn,” Hort Science 9,79 (1974)). When homozygous, the sugary enhancer-1 (se1) alleleincreases total sugar in conventional sugary-1 (su1) sweet corn varietycorn kernels to levels that are comparable to those in shrunken-2 (sh2)sweet corn variety corn kernels, and without a reduction inphytoglycogen content or an appreciable reduction in WSP levels. Thisallele also increases total sugar in conventional shrunken-2 (sh2) sweetcorn variety corn kernels (also without a reduction in phytoglycogencontent or an appreciable reduction in WSP levels).

The effects of the sugary enhancer-1 (se1) gene are corn kernel elevatedtotal sugars, lighter color, and slow dry down, and were originallyobserved in an inbred corn line designated as IL1677a. It was only laterthat these effects were attributed to the sugary enhancer-1 (se1) gene.(Y. Tadmer et al., “RFLP Mapping of the Sugar Enhancer 1 Gene in Maize,”TAG Theoretical and Applied Genetics 91, 489-494 (1995); R A Brink,“Identity and Sources of a Sugary Enhancer Gene Located on the Long Armof Chromosome 4 in Maize,” J. Heredity 82, 176 (1991).)

The sugary enhancer-1 (se1) gene confers a higher moisture content tosweet corn kernels during postharvest periods of time, and alsomaintains relatively increased levels of phytoglycogen during this time.Additional benefits of this gene are reduced kernel pericarp levels(i.e., thickness), rendering corn kernels with an improved tenderness,and elevated levels of the sugar maltose. (J E Ferguson et al.,“Genetics of Sugary Enhancer (Se), an Independent Modifier of Sweet Corn(Su),” J. Heredity 69(6), 377-380 (1978).)

When both the sugary enhancer-1 (se1) gene and the sugary-1 (su1) geneare recessive, the sugary enhancer-1 (se1) gene very advantageouslyconfers from about 1.5 to about 2 times more sucrose to corn kernels attheir peak harvest maturity in comparison with sugary (su1) gene mutantcorn kernels.

The sugary enhancer-1 (se1) gene locus is situated on the long arm ofchromosome 2 in sweet corn. Identifiable variants of the sugaryenhancer-1 (se1) gene are currently being evaluated and characterized.

Apparent difficulties in the genomic characterization of the sugaryenhancer-1 (se1) gene have previously been encountered by scientists.Such difficulties are considered, in part, to be due to its ratherdifficult concomitant phenotypic measurement.

The enzymatic basis for the sugary enhancer-1 (se1) gene currently doesnot appear to be known, and the nucleotide sequence of the sugaryenhancer-1 (se1) gene currently does not appear to be known, and is notpresent in the Maize Genetics and Genomics Database or GenBank database.However, the inheritance of the sugary enhancer-1 (se1) gene can bedetermined by those having ordinary skill in the art by following nearbymolecular markers on chromosome 2, as is described herein. Such adetermination may also be made for other mutant genes.

Additional information about the sugary enhancer-1 (se1) gene is presentin D. R. La Bonte et al., “Sugary Enhancer (se) Gene Located on the LongArm of Chromosome 4 in Maize (Zea mays L.), The Journal of Heredity 82,176-178 (1991); and J. E. Ferguson et al., “The Genetics of SugaryEnhancer (se), an Independent Modifier of Sweet Corn,” The Journal ofHeredity 69(6), 377-380 (1978).

Sh2 (Shrunken-2) Mutant Gene

In 1953, it was suggested that the mutant, recessive shrunken-2 (sh2)gene may have an application in the sweet corn industry. (J. R.Laughnan, “The Effects of sh2 Factor on Carbohydrate Reserves in theMature Endosperm of Maize,” Genetics 38, 485-499 (1953).) Since then, asignificant amount of research has been performed in connection withhigh sugar-content corn and the shrunken-2 (sh2) gene, which is locatedon the long arm of chromosome 3 in sweet corn, and encodes the largesubunit of ADP-glucose pyrophosphorylase (AGP), a Class I enzyme. Thisenzyme is important in the conversion pathway of sucrose to starch.

The conventional shrunken-2 (sh2) class of sweet corns (designated as“supersweet”) comprises the vast majority of the U.S. commercial cornmarket. Mature dry shrunken-2 (sh2) variety corn kernels containapproximately twice the total sugar content, approximately ⅓ to ½ of thestarch levels, and only trace levels of phytoglycogen, in comparisonwith conventional sugary-1 (su1) variety corn kernels. Shrunken-2 (sh2)type sweet corns generally result in dramatically reduced totalcarbohydrate levels at peak maturity, and express approximately two ormore times the sucrose in comparison with conventional sugary-1 (su1)mutant sweet corn. Further, sugar retention at the post prime eatingstage (i.e., during the period of time immediately following the primeeating stage) in these sweet corns is generally significantly extendedrelative to conventional sugary-1 (su1) and sugary enhancer-1 (se1)mutant sweet corns.

It has been determined that including the shrunken-2 (sh2) gene in thegenetic makeup of corn advantageously has an effect of increasing thecorn's total sugar content and, thus, sweetness. It has also beendetermined, however, that including this gene in the genetic makeup ofcorn very disadvantageously significantly lowers the corn's watersoluble polysaccharide (WSP) content, reducing its endosperm content,and lowering its starch content (and its associated energy level).Disadvantageously, conventional shrunken-2 (sh2) varieties of corngenerally lack the smooth and creamy texture of the conventionalsugary-1 (su1) and sugary enhancer-1 (se1) mutant corn varieties as aresult of such decreased levels of water soluble polysaccharides. (J AShultz et al., “Current Models for Starch Synthesis and the SugaryEnhancer 1 (se1) Mutation in Zea mays,” supra.) Also verydisadvantageously, as a result of a reduced starch content, and anassociated reduced level of energy, conventional shrunken-2 (sh2)varieties of corn generally have a significantly reduced germination andseedling vigor, fitness and/or health during germination, seedlingemergence from soil, and plant development and growth in comparison withthe conventional sugary-1 (su1) and sugary enhancer-1 (se1) sweet cornvarieties, resulting in light-weight, thin and spindly-looking,easily-damaged corn plants, which often readily die when confronted withenvironmental or other stresses, potentially causing corn growers tolose entire crops of corn, and the associated potential earnings fromsuch crops. Shrunken-2 (sh2) varieties of sweet corn generally express amarkedly collapsed kernel physical appearance at the dry seed stage, andthis dry seed “shrunken” appearance, and corresponding reduced kernelstarch reserves, tends to render a relatively diminished seedlingemergence and vigor at planting time or during germination. In general,mechanical precision seeding and stand establishment are markedly moredifficult due to the dry seed collapsed and “shrunken” physicalappearance. The germination of such seeds can be problematic both ininbred production and in hybrid stands.

To improve vigor and germination, dent corn (a species of field corn)has been used as the genetic background for the shrunken-2 (sh2) gene.However, the dominant dent corn genes can very disadvantageouslynecessitate an isolation of the shrunken-2 hybrids from both field andsweet corn, and any foreign pollen can cause all of the corn kernels tobe dent corn in character. In addition, shrunken-2 hybrids requirespatial or temporal isolation from sugary-1 (su1) and sugary enhanced-1(se1) sweet corn varieties due to the presence of dominant Su1Su1alleles utilized in the shrunken-2 (sh2) “supersweet” sweet cornvarieties.

Comparison of Sucrose Levels, Sugar Retention Abilities and PericarpLevels

Comparisons of the sucrose levels, sugar retention (holding) abilities,pericarp levels and changes in pericarp levels of conventional sweetcorn varieties containing the sugary-1 (su1) gene, the sugary enhancer-1(se1) gene or the shrunken-2 (sh2) gene at the prime eating stage, orover a seven-day period, are shown in FIGS. 1-4, respectively. (Abbottand Cobb, Inc., Plant Protection No. 9600094 (1998).)

FIG. 1 provides a generalized comparison of representative endospermsucrose levels for the conventional sugary-1 (su1), sugary enhancer-1(se1) and shrunken-2 (sh2) genetic mutant lines at the prime eatingstage (at a level of approximately 75% moisture). FIG. 1 shows that thesugary-1 (su1) line has the lowest level of sucrose (about 7.5%),followed by the sugary enhancer-1 (se1) line (about 12.5%), and then bythe shrunken-2 (sh2) line, which has a much higher level of sucrose incomparison with the other two lines (about 27.5%).

FIG. 2 shows the relative endosperm sugar retention or “holding ability”at room temperature for the conventional sugary-1 (su1), sugaryenhancer-1 (se1) and shrunken-2 (sh2) genetic mutant lines at the primeeating stage (at a level of approximately 75% moisture) over a seven dayinterval (Days 1-7). It shows representative changes in endospermsucrose levels over time for the three different genetic types. FIG. 2shows that the sugary-1 (su1) line has the lowest level of sucrose atall times during the 7-day period (ranging from about 4% on Day 1 toabout 1% on Day 7), followed by the sugary enhancer-1 (se1) line(ranging from about 12% on Day 1 to about 2% on Day 7), and then by theshrunken-2 (sh2) line, which has a much higher level of sucrose overeach of Days 1-7 in comparison with the other two lines (ranging fromabout 25% on Day 1 to about 13% on Day 7).

FIG. 3 provides a comparison of representative pericarp levels for theconventional sugary-1 (su1), sugary enhancer-1 (se1) and shrunken-2(sh2) genetic mutant lines at the prime eating stage (at a level ofapproximately 75% moisture). FIG. 3 shows that the sugary enhancer-1(se1) line has the lowest level of pericarp (about 0.75%), followed bythe sugary-1 (su1) line (about 1.1%), and then by the shrunken-2 (sh2)line, which has a much higher level of pericarp in comparison with theother two lines (about 1.6%).

FIG. 4 shows the relative pericarp levels at room temperature for theconventional sugary-1 (su1), sugary enhancer-1 (se1) and shrunken-2(sh2) genetic mutant lines at the prime eating stage (at a level ofapproximately 75% moisture) over a seven day interval (Days 1-7). Itshows representative changes in pericarp levels over time for the threedifferent genetic types.

FIG. 4 shows that the shrunken-2 (sh2) line generally has the lowestlevel of pericarp during the 7-day period (ranging from about 1.6% onDay 1 to about 2.1% on Day 7), followed by the sugary enhancer-1 (se1)line (ranging from about 1.1% on Day 1 to about 4.9% on Day 7) and thesugary-1 (su1) line (ranging from about 1.7% on Day 1 to about 4.9% onDay 7).

The major advantages of the higher sugar types of corn, i.e., sugaryenhancer-1 (se1) and shrunken-2 (sh2) varieties, are their: (i) greatersweetness; (ii) longer harvest window of time; and (iii) longer shelflife. A higher initial sugar level, and a slower sugar loss at harvest,or during the period of time immediately following the prime eatingstage, provide greater flexibility of harvest, and of handlingconditions, and a longer shelf life for the corn.

Sh2-i (Shrunken-2i) Mutant Gene

U.S. Pat. No. 6,184,438 B1 describes an identification andcharacterization of a mutant form of the shrunken-2 (sh2) gene,designated as shrunken-2i (sh2-i). When present in maize plants, thismutant allele (and variants thereof) is stated to confer enhancedgermination characteristics to these plants as compared to maize plantsthat express the sh2. This patent describes methods for transformingplants with this mutant allele (and variants), and plants that have thismutant allele incorporated into their genomes.

ADP-glucose pyrophosphorylase is a maize endosperm enzyme that is animportant enzyme in the synthesis of starch, and catalyzes a conversionof ATP and α-glucose-1-phosphate to ADP-glucose and pyrophosphate.ADP-glucose arising from the action of this enzyme is the major donor ofglucose for starch biosynthesis in plants. AGP enzymes have beenisolated from plant photosynthetic and non-photosynthetic tissues, andis a heterotetramer that contains two different subunits.

Maize endosperm ADP-glucose pyrophosphorylase is composed of twodissimilar subunits that are encoded by two unlinked genes, shrunken-2(sh2) and brittle-2 (bt2). (M. Bhave et al., “Identification andMolecular Characterization of Shrunken-2 cDNA Clones of Maize,” ThePlant Cell 2:581-588 (1990); J. Bae et al., “Cloning andCharacterization of the Brittle-2 Gene of Maize,” Maydica 35:317-322(1990).) These genes encode the large subunit and the small subunit ofthis enzyme, respectively. The protein produced by the shrunken-2 genehas a predicted molecular weight of 57,179 Da. (J. Shaw et al., “GenomicNucleotide Sequence of a Wild-Type Shrunken-2 Allele of Zea mays,” PlantPhysiol. 98:1214-1216 (1992).)

Shrunken-2 (sh2) and brittle-2 (bt2) maize endosperm mutants generallyhave greatly reduced starch levels, which disadvantageously correspondwith greatly reduced or deficient levels of AGP activity. Mutations ofeither gene have been shown to reduce AGP activity by about 95%. (C.Tsai et al., “Starch-Deficient Maize Mutant Lacking AdenosineDiphosphate Glucose Pyrophosphorylase Activity,” Science 151:341-343(1966); D. Dickinson et al., “Presence of ADP-Glucose Pyrophosphorylasein Shrunken-2 and Brittle-2 Mutants of Maize Endosperm,” Plant Physiol.44:1058-1062 (1969).) It has also been observed that enzymaticactivities increase with the dosage of functional wild type shrunken-2(sh2) and brittle-2 (bt2) alleles, whereas mutant enzymes generally havealtered kinetic properties. AGP appears to be the rate limiting step instarch biosynthesis in plants. (D. Stark et al., “Regulation of theAmount of Starch in Plant Tissues by ADP Glucose Pyrophosphorylase,”Science 258:287-292 (1992).)

The cloning and characterization of the genes encoding the AGP enzymesubunits have been reported for various plants, and include shrunken-2(sh2) cDNA, shrunken-2 (sh2) genomic DNA, and brittle-2 (bt2) cDNA frommaize, small subunit cDNA and genomic DNA from rice, small and largesubunit cDNAs from spinach leaf, and potato tuber. In addition, cDNAclones have been isolated from wheat endosperm and leaf tissue andArabidopsis thaliana leaf.

Gene splicing is essentially a two-step cleavage-ligation reaction thatcan produce molecular lesions in genes, such as the wildtype shrunken-2(sh2) gene. The first step involves the cleavage at the 5′ splice sitethat leads to the formation of an intron lariat with the adenosineresidue of the branch point sequence located upstream to the 3′ splicesite. This is followed by the ligation of the exon and release of theintron lariat. (M. J. Moore et al., “Evidence of Two Active Sites in theSpliceosome Provided by Stereochemistry of Pre-mRNA Splicing,” Nature365:364-368 (1993); M. J. Moore et al., “Splicing of Precursors to mRNAsby the Spliceosome” in The RNA World. 303-308 (R. Gesteland and J.Atkins, eds., Cold Spring Harbor Laboratory Press, 1993); P. A. Sharp,“Split Genes and RNA Splicing,” Cell 77:805-815 (1994); J. W. S. Brown,“Arabidopsis Intron Mutations and Pre-mRNA Splicing,” Plant J.10(5):771-780 (1996); G. G. Simpson et al., “Mutation of PutativeBranchpoint Consensus Sequences in Plant Introns Reduces SplicingEfficiency,” Plant J. 9(3):369-380 (1996); G. G. Simpson et al.,“Splicing of Precursors to mRNA in Higher Plants: Mechanism, Regulationand Sub-nuclear Organization of the Spliceosomal Machinery,” Plant Mol.Biol. 32:1-41 (1996).) This set of events is carried out by pre-mRNAassociation with a conglomeration of small nuclear RNA (snRNAs) andnuclear proteins that forms a dynamic large ribonucleosome proteincomplex (a spliceosome). This fundamental process, common to alleukaryotic gene expression, can have a diverse impact on the regulationof gene expression. For example, imprecise or inaccurate pre-mRNAsplicing often imparts a mutant phenotype, whereas alternative splicingis sometimes important in the regulation of gene expression. (C. F. Weilet al., “The Effects of Plant Transposable Element Insertion onTranscription Initiation and RNA Processing,” Annu. Rev. Plant Physiol.Plant Mol. Biol. 41:527-552 (1990); R. Nishihama et al., “PossibleInvolvement of Differential Splicing in Regulation of the Activity ofArabidopsis ANP1 that is Related to Mitogen-Activated Protein KinaseKinase Kinases (MAPKKKs),” Plant J. 12(1):39-48 (1997); M. Golovkin etal., “Structure and Expression of a Plant U1 snRNP 70K Gene: AlternativeSplicing of U1 snRNP 70K Pre-mRNAs Produces Two Different Transcripts,”Plant Cell 8:1421-1435 (1996); J. Callis et al., “Introns Increase GeneExpression in Cultured Maize Cells,” Genes and Development 1:1183-1200). There are structural/sequence differences that maydifferentiate plant introns from those of vertebrate and yeast introns.(G. J. Goodall et al., “The AU-Rich Sequences Present in the Introns ofPlant Nuclear Pre-mRNAs Are Required for Splicing,” Cell 58:473-483(1989); G. J. Goodall et al., “Different Effects of Intron NucleotideComposition and Secondary Structure on Pre-mRNA Splicing in Monocot andDicot Plants,” EMBO J. 10(9):2635-2644 (1991).) One feature thatdistinguishes plant introns from those of other organisms is their AUrichness. This has been implicated to be essential for intronprocessing, and for a definition of the intron/exon junction. (H. Lou etal., “3′ Splice Site Selection in Dicot Plant Nuclei is PositionDependent,” Mol. Cell. Biol. 13(8):4485-4493 (1993); A. J. McCullough,“Factors Affecting Authentic 5′ Splice Site Selection in Plant Nuclei,”Mol. Cell. Biol. 13(3):1323-1331 (1993); J. C. Carle-Urioste et al., “InVivo Analysis of Intron Processing using Splicing-Dependent ReporterGene Assays,” Plant Mol. Biol. 26:1785-1795 (1994).) The requirement ofan AU rich region appears to be more stringent in dicots in comparisonwith monocots, and some monocot introns are GC-rich.

The wildtype shrunken-2 (sh2) gene, the nucleotide sequence of which isshown in SEQ ID NO. 1, has 16 exons which, in term, are separated by 15introns, as is shown in FIG. 5. This gene is estimated to beapproximately 6000 base pairs long. One intron, in particular, is ofsignificance for the sh2-i allele. This intron, designated “intron 2,”contains at least about 7,800 base pairs in the sh2-i gene.

In comparison with the wildtype shrunken-2 (sh2) gene, the mutantshrunken-2i (sh2-i) gene is characterized by a single base pair changeat the end of intron 2, as is shown in FIGS. 5 and 9. The mutantpolynucleotide comprises a substitution of the wild-type terminal baseat the end of intron 2 from a G to another base, such as to A, C or T(and not the wild type G nucleotide), and preferably to A. For example,if the shrunken-2 gene of maize contains a G to A mutation of theterminal nucleotide of this intron, the result would be a change of theAG nucleotide sequence that is found at the terminus of this plant geneintron to an AA sequence at the 3′-terminus of this intron. In otherwords, the result is a molecular lesion of the sh2-i allele in which ithas undergone a G to A mutation at the terminal base of intron 2 in themaize sh2 gene.

The mutant sh2-i allele (and variants thereof), when expressed in aplant, such as maize, provides the plant with enhanced growthcharacteristics, such as an enhanced germination, and an enhancedseedling, seed and/or plant vigor, in comparison with other sweet cornvarieties, in combination with desirable consumer traits, such assweetness.

Inbred sweet corn lines containing the mutant sh2-i allele (“sh2-iconversion inbreds”) generally demonstrate sweetness, and sugar levels,that are similar to conventional shrunken-2 (sh2) counterparts at thepeak eating stage (at approximately 75% moisture). However, at a pointin time just past the prime eating stage, mutant sh2-i inbred plantlines initiate a rapid acceleration of starch synthesis. This results indry seed phenotypes that are physically significantly fuller and heavierthan their shrunken-2 (sh2) counterparts and, to some degree, resemble amodified flint corn seed appearance. The net result is an overallenhancement of seed and seedling germination and vigor, and plant vigor,along with associated enhanced plant growth characteristics. Thisimproved germination, and accelerated plant growth phenomenon, directlyresults in improved varietal crop yield potentials and consistencies.

Laboratory cold soil germination testing conducted by the presentinventor comparing conventional shrunken-2 (sh2) hybrid maize varietieswith maize varieties expressing the shrunken-2i (sh2-i) gene resulted insubstantially stronger scores for the sh2-i hybrids. Table 2 belowprovides laboratory cold soil germination scores for corn hybrid nearisogenic lines (NILs) designated as ACX 5137Y (not expressing the sh2-iallele) and ACX SS 7501Y (expressing the sh2-i allele). These areessentially comparisons of two maize hybrids differing only in thepresence of the sh2-i allele. Scores represent means of three replicatedtests of 100 kernels each.

TABLE 2 Cold Soil Germination Scores for Isoline Hybrids Cold SoilGermination Isoline Hybrid (% Germination) ACX 5137Y (not containing thesh2-i 82 allele in its genome) ACX SS 7501Y (containing the sh2-i 97allele in its genome)

Similar laboratory results have been generated for numerous other hybridcomparisons between conventional shrunken-2 (sh2) hybrids containing, ornot containing, the sh2-i allele. In addition, field germination andstand establishment data in Florida and California have substantiatedlaboratory data findings.

Organoleptic testing of numerous corn hybrid near isogenic lines (NILs)(conventional shrunken-2 (sh2) hybrid backcross conversions containing,and not containing, the sh2-i allele), however, disadvantageouslyyielded sweetness evaluation scores indicating a rapid starch buildup inthe sh2-i hybrids, generally immediately following the prime eatingstage. The sh2-i hybrids exhibited reduced sweetness within about 24 toabout 48 hours immediately following the prime eating stage, with acorresponding significant sugar degradation and loss at about three dayspost prime eating stage.

FIG. 6 presents mean organoleptic averages for starch accumulation forconventional shrunken-2 (sh2) hybrid near isogenic lines (NILs) that donot contain the mutant shrunken-2i (sh2-i) allele in comparison withthose for conventional shrunken-2 (sh2) hybrid corresponding nearisogenic lines (NILs) that contain the mutant shrunken-2i (sh2-i) alleleover time in Days 1-7 immediately following the prime eating stage (at alevel of approximately 75% moisture). The organoleptic scores range from1 (sweet with little or no starch taste) to 10 (very little sweetnesswith a considerable starch taste). FIG. 6 shows that the conventionalshrunken-2 (sh2) hybrid near isogenic lines (NILs) that do not containthe mutant shrunken-2i (sh2-i) allele have significantly lowerorganoleptic scores (ranging from about 1 on Day 1 to about 4 on Day 7)in comparison with the conventional shrunken-2 (sh2) hybridcorresponding near isogenic lines (NILs) that contain the mutantshrunken-2i (sh2-i) allele (ranging from about 1.9 on Day 1 to about 7.9on Day 7).

In essence, the incorporation of the shrunken-2i (sh2-i) allele intoconventional sweet corn varieties is considered to be impractical due toa rapid accumulation of starch in the period of time immediatelyfollowing the prime eating state, and the associated loss of holdingability and shelf life.

DESCRIPTION OF OTHER ART

U.S. Pat. No. 3,971,161 describes methods that are stated to producehybrid sweet corn seeds in commercial quantities, increase the sugarcontent of sweet corn without seriously reducing the water solublepolysaccharide content, and produce seeds that provide a sweet cornsuitable for processing with a minimal amount of extraneous sweeteners.It states that the sugar content of sweet corn can be increased withoutseriously reducing the water soluble polysaccharide by using theshrunken-2 (sh2) gene, and that combining a sweet corn which is ahomozygous sugary-1 (su1) inbred with a sweet corn (homozygous su1 sh2)inbred will result in a heterozygous hybrid that has approximately 50%more sucrose, 33% more total sugars and a water soluble polysaccharidelevel near that of sweet corn (homozygous su 1). The high water solublepolysaccharide and sucrose levels are stated to be particularlydesirable in food processing industries, such as the canning andfreezing industries.

U.S. Pat. Nos. 5,589,618 and 5,650,557 describe a variant of the maizegene shrunken-2 (sh2) that is designated Sh2-m1Rev6, and a method ofusing that gene. Sh2-m1Rev6 is stated to encode a subunit of theADP-glucose pyrophosphorylase (AGP) enzyme that has additional aminoacids inserted in, or near, the allosteric binding site of the protein.Corn seed expressing the Sh2-m1Rev6 gene is stated to have a 15% weightincrease over wild type seed, and the increase in seed weight is statednot to be associated simply with an increase in percentage starchcontent of the seed.

U.S. Pat. No. 5,746,023 describes a method for identifying geneticmarkers that are stated to be linked to alleles conferring yieldpotential of a crop species. By conducting genetic marker analysis of aset of current elite lines, and the ancestral population from which theywere derived by decades of plant breeding, the '023 patent states thatone may determine and compare the expected, and observed, allelefrequencies within elite populations at numerous polymorphic loci.

U.S. Pat. No. 5,912,413 describes the starch debranching enzyme encodedby the sugary (su1) gene that is active in maize (Zea mays) endosperm,and the cDNA and gene sequences encoding this enzyme. The amino acidsequence of the enzyme is stated to be significantly similar to that ofbacterial isoamylases, enzymes that hydrolyze α-(1→46) glycosidic bonds.This patent states that amino acid sequence similarity establishes su1as a member of the α-amylase super family of starch hydrolytic enzymes.The '413 patent also discloses antibodies that are reactive with the su1protein, methods of producing antibodies to the su1 protein, methods ofproducing fusion proteins including su1, and methods of producingtransgenic plants with a modified su1 gene.

U.S. Pat. No. 6,184,438 B1 describes mutant alleles of the genes thatencode the large subunit of AGP-glucose pyrophosphorylase in plants,methods for transforming plants with the mutant alleles, and plants thathave the mutant alleles incorporated into their genome. When present inmaize plants, the mutant alleles are stated to confer enhancedgermination characteristics to the plants, as compared to plants thatexpress the sh2-R genes.

U.S. Pat. No. 6,756,524 B2 describes an isolation and identification ofa nucleic acid molecule that is stated to regulate fruit size and/orcell division in plants, and the protein encoded by this nucleic acidmolecule. The '524 patent also describes an expression vector containingthe encoding nucleic acid, and methods whereby fruit size is stated tobe reduced and/or increased, and cell division is stated to beregulated, by a transformation of plants with this nucleic acidmolecule. It also discusses host cells, transgenic plants and plantseeds containing this nucleic acid molecule.

U.S. Pat. No. 7,084,320 B2 describes methods for selecting a parentinbred plant line (having a particular genetic background) that has agood combining ability, for example, for the production of hybrid plantlines, from a collection of parental lines. Such a parent inbred plantline is referred to as being “an excellent combiner” in breedingexperiments that are described in this patent. Upon crossing of suchparent inbred plant line with another parent inbred plant line (having adifferent genetic background), the two parent inbred plant lines arestated to be capable of yielding a hybrid plant line with high heterosiseffect, and the seeds from the crossed selected inbred lines arecollected and, optionally, planted and grown to obtain the hybridplants. The '320 patent also describes methods for determining theagronomical performance of different plant lines, including theforegoing hybrids, which it states can be performed in vitro bydetermining the electron flow in mitochondria under control and stressconditions. The '320 patent describes an object as being to provide amethod for selecting a hybrid (or other) plant line having the highestgrowth and yield vigor from a collection of plant lines from the samespecies (variety). It describes and shows (FIG. 1) a vigor assay that itstates can be used to identify plant lines that are affected in theirvigor.

M. Clancy and L C Hannah, “The Mutations sh2-i and sh2-N2340 Share anIdentical Intron Splice Site Mutation and are Most Likely the SameAllele,” Maize Genetics Cooperation Newsletter 80 (2006), states thatthe mutant alleles sh2-i and sh2-N2340, which are publicly availablefrom the Maize Stock Center (Urbana/Champaign, Ill.), were generatedusing EMS mutagenesis, and condition an intermediate or leaky phenotype.It further states that sh2-i and sh2-N2340 kernels are visually similar,trace to the same source, and are less severely collapsed when mature incomparison with an sh2-R reference allele. It states that sequencingestablished that sh2-i contains a G to A transition at the 3-terminus ofintron 2 (Lal et al., Plant Physiol. 120:65-72, 1999), and thatapproximately 10% of sh2-i transcripts are correctly spliced utilizingthe mutant intron splice site, generating a low level of adenosinediphosphate glucose pyrophosphorylase activity that results in theintermediate kernel phenotype. It states that, in order to determinewhether or not the alleles sh2-i and sh2-N2340 contain the samemutation, young shoot material was harvested from germinating sh2-N2340kernels, genomic DNA was prepared using Plant DNAZOL Reagent(Invitrogen, Carlsbad, Calif.), and DNA spanning exons 1 through 4 wasamplified via PCR using the primers described by Lal et al. It statesthat, because sequencing of the PCR product established that sh2-i andsh2-N2340 share the same G to A transition of the final nucleotide inintron 2, it appears that the same mutation bears two differentdesignations (i.e., there are two different names for the same mutantallele).

Although the shrunken-2i (sh2-i), sugary-1 (su1) and sugary enhancer-1(se1) genes are known, no one to date has been able to successfullycombine these genes to produce plants, plant materials and seeds havingan enhanced vigor, while also retaining desirable consumer traits, suchas an elevated sugar level during the period following the prime eatingstage, and this has led to frustration among plant breeders, plantgrowers and consumers. There has, thus, been a long-felt, butunresolved, need in the industry for cost-effective, reliable, efficientand successful methods for commercially producing inbred and hybridplants having seeds that exhibit a fuller content (i.e., more storedenergy) at the dry seed stage in comparison with conventionalcounterpart plants, resulting in an enhanced vigor during seedgermination, seedling emergence from the soil and plant development(like conventional sh2-i sweet corn), but that also retain desirableconsumer traits, such as an elevated sugar level during the periodfollowing the prime eating stage, maintaining a sweet or desirableflavor of the plant, or its plant parts (seeds, fruits, vegetables, cornkernels, ears of corn, or the like). Improved growth and tastecharacteristics of plants, such as sweet corn, would confer asignificant advancement in the commercial production of these plants.

Various commercial plant breeders and growers, such as Rogers NK (Boise,Id.) and Syngenta Seeds, Inc. (Stanton, Minn.), have tried, and failed,to successfully combine traits to achieve a plant variety that ispleasing to both producers and consumers. While Rogers NK has onecommercial maize product (named “Brighton”) that includes the maizeshrunken-2i (sh2-i) allele in its genome, this product has not beencommercially successful because the eating quality (taste, texture andthe like) of this product is not desirable to consumers. Because thisproduct does not taste good (i.e., it has a starchy, unsweet taste), ithas been very undesirable to corn growers and gardeners who are plantingsweet corn for consumer acceptance.

Further, none of the above references, or others that are describedherein, teach or suggest the methods, plants, plant materials or seedsof the present invention.

SUMMARY OF THE INVENTION

The present invention provides unique, cost-effective, reliable,efficient and successful methods for developing and producing plants,plant materials and seeds, such as sweet corn kernels, and sweet corn,that very advantageously receive, and have, multiple very desirableattributes for consumers of these products, as well as for commercialplant growers and home gardeners, and to improved plants, plantmaterials and seeds that are produced in accordance with these methods.These inventive methods provide novel hybrid plants, plant materials andseeds having the mutant shrunken-2i (sh2-i) allele incorporated intotheir genomes, preferably sequentially along with one or more othermutant alleles, such as the sugary-1 (su1), sugary enhancer-1 (se1)and/or shrunken-2 (sh2) alleles, and that very advantageously havemultiple very beneficial and desirable characteristics, generallyincluding a smooth and creamy texture, an enhanced sugar level thatresults in a very desirable sweet or other taste, an extended sugarretention ability (and associated taste benefits, such as the sweettaste of sweet corn) at the post prime eating stage, a longer harvestwindow of the plant, a longer holding ability of the plant (ears ofsweet corn and the like), and a longer shelf life of the plant beforesweetness deteriorates after the prime eating stage, seeds and/orkernels that physically are fuller, and have a higher carbohydrate andwater soluble polysaccharides (WSP) content, at the dry seed stage,and/or significantly enhanced vigor and fitness to the plant, plantmaterial and/or seed during seed germination, seedling emergence fromsoil, and plant development.

The present invention also provides methods for incorporating the mutantsh2-i gene into the genomes of commercially acceptable (and other) sweetcorn female (and other) parent lines not originally including the sh2-igene in their genomes, and for identifying female (and other) sweet cornparent lines into which the mutant sh2-i gene has successfully beenincorporated (often referred to herein as “sh2-i conversion lines”).

Further, the present invention provides unique and successfulconstruction processes for producing hybrid sweet corn (and other plant)lines having a series of multiple very desirable traits, as aredescribed in detail herein, which are optionally, but preferably,developed using the following types of female and male sweet corn (orother plant) parent lines:

-   -   (i) a female sweet corn (or other plant) parent line containing        the mutant sh2-i endosperm allele in its genome (i.e., an “sh2-i        conversion line”):        -   preferably as a conversion of a conventional “supersweet”            female parent line including the allelic combination of            Su1Su1 sh2sh2 in its genome, optionally, but preferably,            using molecular markers and sequentially layering the mutant            sh2-i allele against a genetic background (genotype) of            Su1Su1 sh2sh2 to produce, for example, a female “Su1Su1            sh2-i sh2-i conversion line” including the desired endosperm            mutant allele genotype Su1Su1 sh2-i sh2-i in its genome, and            produced by incorporating the mutant sh2-i endosperm gene            into a conventional “supersweet” female parent Su1Su1 sh2sh2            line (i.e., including an Su1Su1 sh2sh2 genetic background or            genotype); and        -   more preferably as a conversion of a female parent line            including the double homozygous recessive allelic            combination of su1su1 se1se1 in its genome, such as su1su1            se1se1 or su1su1 se1se1 sh2sh2, optionally, but preferably,            using molecular markers and sequentially layering the mutant            sh2-i allele against a genetic background (genotype) of the            homozygous recessive se1se1 and su1su1 alleles to produce,            for example, a female “su1su1 se1se1 sh2-i sh2-i conversion            line” including the desired endosperm mutant allele genotype            su1su1 se1se1 sh2-i sh2-i in its genome, and produced by            incorporating the mutant sh2-i endosperm gene into a female            plant line including a homozygous recessive genotype of            su1su1 se1se1 in its genome (i.e., including an su1su1            se1se1 or su1su1 se1se1 sh2sh2 genetic background or            genotype); and    -   (ii) a male sweet corn (or other plant) parent line including in        its genome a single, double or triple homozygous recessive        allelic construction or combination in connection with the        sugary (su 1), sugary enhancer-1 (se1) and/or shrunken-2 (sh2)        mutant endosperm (or other) alleles, such as the genotype Su1Su1        Se1Se1 sh2sh2 (single construction), and preferably the double        homozygous recessive allelic combination su1su1 Se1 Se1 sh2sh2        or su1su1 sh2sh2, and even more preferably the triple homozygous        recessive allelic combination su1su1 se1se1 sh2sh2 (with both        such double and triple homozygous recessive endosperm mutant        allelic combinations including the double homozygous recessive        endosperm mutant su1su1 and sh2sh2 alleles).        The above female parent sweet corn (or other plant) lines        express, and pass down to hybrids produced therewith (when        constructed in the manner that is described herein), the very        desirable traits of an enhanced seedling germination and an        enhanced seedling vigor. The above male parent sweet corn (or        other plant) lines, on the other hand, express, and pass down to        hybrids produced therewith (when constructed in the manner that        is described herein), the very desirable grower and/or consumer        traits of producing sweet corn kernels (or other plant parts)        that: (i) are exceptionally high in sugar (sucrose) content and,        as a result, have a very sweet taste; (ii) have very tender        pericarps; and (iii) have significant holding abilities        (retention of higher sugars and tender kernel pericarps). These        male parent traits are believed to be a result of the double        recessive homozygous su1su1 and sh2sh2 genotypic endosperm        mutant allelic combinations that are preferably present in both        the above double and triple recessive allelic combinations of        the male parent lines (double: su1su1 Se1Se1 sh2sh2 or su1su1        sh2sh2; triple: su1su1 se1se1 sh2sh2).

The above female and male parent sweet corn (or other plant) line mutantendosperm alleles, when expressed in such a manner in a sweet corn (orother plant) hybrid, very advantageously provide the hybrid withsignificantly enhanced growth characteristics, such as an enhancedseedling germination and an enhanced seedling vigor, in comparison withvery similar or other sweet corn (or other plant) hybrids that do nothave such mutant endosperm alleles expressed in this manner (i.e., thatwere not developed using such female and male parental lines). This isvery apparent from the comparison testing data that is set forth in the“Examples” section hereinbelow, for example, as a result of testing inboth the laboratory and in the field, and of organoleptic testing, oftwo different parent sweet corn lines or hybrid sweet corn lines thatare identical, with the one exception that one includes the sh2-iendosperm mutant allele in its genome, and the other one does not.

The present invention also provides an identification and incorporationof the mutant sh2-i endosperm gene into commercially acceptable (orother) sweet corn (or other plant) parental lines to produce hybridplants, plant materials and seeds using a combination of: (i) femaleparental lines including in their genome the shrunken-2i (sh2-i) mutantendosperm allele preferably incorporated into a conventional“supersweet” (Su1Su1 sh2sh2) genetic background (genotype) to providethe resulting genotype Su1Su1 sh2-i sh2-i, which is characteristic ofenhanced seedling germination and enhanced seedling vigor; and (ii) maleparental lines including in their genome the double (su1su1 Se1Se1sh2sh2 or su1su1 sh2sh2) or triple (su1su1 se1 se1 sh2 sh2) (or other)homozygous recessive mutant endosperm genotypes, which arecharacteristic of being exceptionally high in sugar, thereby providing asweet taste to the products, with very tender kernel pericarps. Theresulting hybrid products have multiple beneficial traits, including asignificantly enhanced vigor and fitness to the plant, plant materialand/or seed during germination, seedling emergence from the soil, andplant development. This benefit is coupled with adequate sugarretention, product holding ability and shelf life at peak eating stage,for example, at approximately 75% kernel moisture, and through at leasta 48-hour harvest window (and often longer).

In one aspect, the present invention provides a method for producing ahybrid plant, plant material or seed that has an enhanced vigor incomparison with a conventional mutant shrunken-2 (sh-2) or mutantshrunken-2i (sh2-i) hybrid plant, plant material or seed, and anenhanced ability to retain sugar over a period of time immediatelyfollowing a prime eating stage thereof, in comparison with aconventional mutant sugary-1 (su1) or shrunken-2i (sh2-i) hybrid plant,plant material or seed, comprising optionally using one or moremolecular markers to preferably, but optionally, sequentially include inthe genome of the plant, plant material or seed a mutant shrunken-2i(sh2-i) endosperm allele and one or more other mutant endosperm allelesthat confer the foregoing characteristics to the plant, plant materialor seed. Although such use of molecular markers in connection with suchsequential layering of mutant endosperm alleles in the genome of theplant, plant material or seed, are both very desirable, and arepreferable, as is discussed herein in detail, particularly in connectionwith Example 4 and Example 8 hereinbelow, such process step is optional.

In another aspect, the present invention provides a method for producinga hybrid plant, plant, plant material or seed that has both an enhancedvigor in comparison with a conventional mutant shrunken-2 (sh-2) orshrunken-2i (sh2-i) hybrid plant, plant, plant material or seed, and anenhanced ability to retain sugar over a period of time immediatelyfollowing a prime eating stage thereof (or one or more other desirabletraits), in comparison with a conventional mutant sugary-1 (su1) orshrunken-2i (sh2-i) hybrid plant, plant, plant material or seed,comprising the following steps in any suitable order:

-   -   (a) identifying an inbred plant line that includes one or more        desired mutant alleles in its genome, singly or in combination,        including, but not limited to, the sugary 1 (su1), sugary        enhancer-1 (se1) and shrunken 2 (sh2) mutant alleles, optionally        using molecular markers;    -   (b) constructing one or more female near isogenic plant lines        (NILs) having a desired genotype, including, but not limited to        the following triple mutant endosperm allelic combinations:        -   (i) Su1Su1 Se1Se1 sh2sh2;        -   (ii) Su1Su1 se1se1 sh2sh2; or        -   (iii) su1su1 se1se1 sh2sh2 (a triple homozygous recessive            mutant endosperm allelic combination);    -   for use as a genetic background (genotype) in a combination with        a female parental plant line that has a shrunken-2i (sh2-i)        mutant allele in its genome;    -   (c) optionally, incorporating a shrunken-2i (sh2-i) mutant        allele into the genome of the female near isogenic plant line        having a desired genetic background of step (b), for example        including a genotype of su1su1 se1se1 sh2sh2 or su1su1 se1se1,        optionally, using one or more molecular markers;    -   (d) selecting a female near isogenic plant line of step (c)        having a shrunken-2i (sh2-i) allele layered over a genetic        background (genotype) of su1su1 se1se1, or of one or more other        desirable mutant alleles, individually or in combination, in its        genome (a female “converted near isogenic line”);    -   (e) optionally, crossing the selected female converted near        isogenic plant line of step (d) with a male plant line including        a triple homozygous recessive mutant endosperm allelic        construction in its genome, wherein such homozygous recessive        mutant endosperm allelic construction is su1su1 se1se1 sh2sh2        (or some other triple homozygous recessive allelic construction        that can provide a hybrid plant with a high or enhanced eating        quality) in its genome to produce a hybrid plant having one or        more desired grower and/or consumer traits;    -   (f) optionally, examining a physical appearance (phenotype) of        seeds (or kernels) resulting from the plants of step (d) and/or        step (e) for characteristics such as smoothness, fullness and/or        relative weight (in comparison with seeds or kernels from        conventional or other plants);    -   (g) optionally, conducting warm, cold and/or other laboratory        and/or field germinations of seeds (or kernels) resulting from        the plants of step (d) and/or step (e) to verify that such seeds        have one or more desired consumer and/or grower traits, such as        an enhanced germination, seedling emergence and plant growth        performance, and vigor, that is associated with a mutant        shrunken-2i (sh2-i) allele; and    -   (h) optionally, conducting one or more organoleptic taste,        pericarp tenderness and/or other tests on plants (or parts        thereof, such as ears of corn) that are grown from seeds (or        kernels) produced by plants of step (d) and/or step (e) to        determine their taste, pericarp tenderness and/or other        organoleptic characteristics, optimally examining the overall        physical and horticultural traits (i.e., phenotype) that are        consistent with plants that express one or more desired grower        and/or consumer traits.        By following the above processes, a regulation of carbohydrate        accumulation and pericarp tenderness (among other traits) can be        manipulated in a plant, plant material and/or seed, and a mutant        shrunken-2i (sh2-i) allele can be incorporated into the genome        of the plant, plant material or seed to give it the desired        production and consumer traits. Plants, plant materials and        seeds can be grown that exhibit a fuller content at the dry seed        stage in comparison with other plant varieties or lines, and        result in an enhanced vigor during seed germination, seedling        emergence from the soil and/or plant development, and that also        maintain an elevated sugar level, resulting in a sweet or other        desirable flavor of the plant, plant material and/or seed over a        period of time immediately following the prime eating stage        thereof. Those having ordinary skill in the art may determine        the number of times that a particular step in the above process,        such as the backcrossing of near isogenic lines, should or must        be performed in order to achieve a successful result of the        process. As is indicated above, some of these steps are        preferably performed multiple times.

In another aspect, the present invention provides a hybrid plant, plant,plant material or seed that has an enhanced vigor in comparison with aconventional mutant shrunken-2 (sh-2) or shrunken-2i (sh2-i) hybridplant, plant, plant material or seed, and an enhanced ability to retainsugar over a period of time immediately following a prime eating stagethereof, in comparison with a conventional mutant sugary-1 (su 1) orshrunken-2i (sh2-i) hybrid plant, plant, plant material or seed,consisting of steps (a) through (h) above, in any suitable order.

In yet another aspect, the present invention provides a plant seed thatis produced by any one of the methods that is described above (orotherwise herein).

In another aspect, the present invention provides a hybrid or otherplant seed comprising a genome including the shrunken-2i (sh2-i) allelethat is sequentially layered across a genetic background of one or moreadditional mutant alleles providing the plant seed with one or moredesirable traits, wherein the plant seed is conferred with an enhancedvigor in comparison with a conventional mutant shrunken-2 (sh-2) orshrunken-2i (sh2-i) plant seed, and an enhanced ability to retain sugarover a period of time immediately following a prime eating stagethereof, in comparison with a conventional mutant sugary-1 (su1) orshrunken-2i (sh2-i) plant seed.

In still another aspect, the present invention provides a hybrid orother plant seed consisting of a genome including the shrunken-2i(sh2-i) allele that is sequentially layered across a genetic backgroundof one or more additional mutant alleles providing the plant seed withone or more desirable traits, wherein the plant seed is conferred withan enhanced vigor in comparison with a conventional mutant shrunken-2(sh-2) or shrunken-2i (sh2-i) plant seed, and an enhanced ability toretain sugar over a period of time immediately following a prime eatingstage thereof, in comparison with a conventional mutant sugary-1 (su1)or shrunken-2i (sh2-i) plant seed.

In another aspect, the present invention provides a plant or plantmaterial that is produced by any one of the methods that is describedabove (or otherwise herein).

In yet another aspect, the present invention provides a hybrid or otherplant or plant material comprising a genome including the shrunken-2i(sh2-i) allele that is sequentially layered across a genetic backgroundof one or more additional mutant alleles providing the plant or plantmaterial with one or more desirable traits, wherein the plant or plantmaterial is conferred with an enhanced vigor in comparison with aconventional mutant shrunken-2 (sh-2) or shrunken-2i (sh2-i) plant orplant material and an enhanced ability to retain sugar over a period oftime immediately following a prime eating stage thereof, in comparisonwith a conventional mutant sugary-1 (su1) or shrunken-2i (sh2-i) plantor plant material.

In another aspect, the present invention provides a hybrid or otherplant or plant material consisting of a genome including the shrunken-2i(sh2-i) allele that is sequentially layered across a genetic backgroundof one or more additional mutant alleles providing the plant or plantmaterial with one or more desirable traits, wherein the plant or plantmaterial is conferred with an enhanced vigor in comparison with aconventional mutant shrunken-2 (sh-2) or a conventional mutantshrunken-2i (sh2-i) plant or plant material and an enhanced ability toretain sugar over a period of time immediately following a prime eatingstage thereof, in comparison with a conventional mutant sugary-1 (su1)or conventional shrunken-2i (sh2-i) plant or plant material.

In yet another aspect, the present invention provides a method forproducing a hybrid plant, plant, plant material or seed that has both anenhanced vigor in comparison with a conventional mutant shrunken-2(sh-2) or shrunken-2i (sh2-i) hybrid plant, plant, plant material orseed, and an enhanced ability to retain sugar over a period of timeimmediately following a prime eating stage thereof (or one or more otherdesirable traits), in comparison with a conventional mutant sugary-1(su 1) or shrunken-2i (sh2-i) hybrid plant, plant, plant material orseed, comprising the following steps in any suitable order:

-   -   (a) identifying an inbred plant line that includes one or more        desired mutant alleles in its genome, singly or in combination,        including, but not limited to, the sugary 1 (su1), sugary        enhancer-1 (se1) and shrunken 2 (sh2) mutant endosperm alleles,        optionally using molecular markers;    -   (b) constructing one or more female near isogenic plant lines        (NILs) having a desired genotype, including, but not limited to        the following triple allelic combinations:        -   (i) Su1Su1 Se1Se1 sh2sh2;        -   (ii) Su1Su1se1se1 sh2sh2; or        -   (iii) su1su1 se1se1 sh2sh2 (a triple homozygous recessive            mutant endosperm allelic combination);    -   for use as a genetic background (genotype) in a combination with        a female parental plant line that includes a shrunken-2i (sh2-i)        mutant allele in its genome;    -   (c) incorporating a shrunken-2i (sh2-i) mutant allele into the        genome of the female near isogenic plant line having a desired        genetic background (genotype) of step (b), optionally, using one        or more molecular markers, wherein the desired genetic        background is su1su1 se1se1 sh2sh2, su1su1 se1se1, Su1Su1se1se1        sh2sh2, Su1Su1 se1se1, Su1Su1 sh2sh2 or another desirable        genetic background;    -   (d) selecting a female near isogenic plant line of step (c)        having a mutant shrunken-2i (sh2-i) allele incorporated into a        genetic background (genotype) of Su1Su1 sh2 sh2, or of one or        more other desirable mutant alleles, individually or in        combination, wherein the female near isogenic plant line        includes an endosperm allelic combination of Su1Su1 sh2-i sh2-i,        or another desirable endosperm allelic combination, in its        genome (a female “converted near isogenic line”), which is        characteristic of an enhanced seedling germination and vigor;    -   (e) optionally, crossing the selected female converted near        isogenic plant line of step (d) with a male plant line including        a homozygous recessive endosperm allelic construction in its        genome, wherein such homozygous recessive endosperm allelic        construction is Su1Su1Se1Se1 sh2sh2, su1su1 Se1Se1 sh2 sh2,        su1su1 se1se1 sh2 sh2, or another homozygous recessive endosperm        allelic combination that can provide a hybrid plant with a high        or enhanced eating quality, to produce a hybrid plant having one        or more desired grower traits, consumer traits, or both traits;    -   (f) optionally, examining a physical appearance (phenotype) of        seeds (or kernels) resulting from the plants of step (d) and/or        step (e) for characteristics such as smoothness, fullness and/or        relative weight (in comparison with seeds or kernels from        conventional or other plants);    -   (g) optionally, conducting one or more warm, cold, or both warm        and cold, laboratory, field, or laboratory and field,        germinations of seeds (or kernels) produced by the plants of        step (d) and/or step (e) to verify that such seeds have one or        more desired consumer and/or grower traits, such as an enhanced        germination, seedling emergence and plant growth performance,        and vigor, that is associated with a mutant shrunken-2i (sh2-i)        allele, or a combination thereof; and    -   (h) optionally, conducting one or more organoleptic taste,        pericarp tenderness and/or other tests on plants, on plant parts        (such as ears of corn), or a combination thereof, that are grown        from seeds (or kernels) produced by the plants of step (d)        and/or step (e) to determine their taste, pericarp tenderness        and/or other organoleptic characteristics, optimally examining        the overall physical and horticultural traits (i.e., phenotype)        that are consistent with plants that express one or more desired        grower and/or consumer traits.

As with the other processes that are described hereinabove, by followingthe process directly above, a regulation of carbohydrate accumulationand pericarp tenderness (among other desirable grower and/or consumertraits) can be manipulated in a plant, plant material and/or seed, and amutant shrunken-2i (sh2-i) allele can be incorporated into the genome ofthe plant, plant material or seed to give it the desired production andconsumer traits. Plants, plant materials and seeds can be grown thatexhibit a fuller content at the dry seed stage in comparison with otherplant varieties or lines, and result in an enhanced vigor during seedgermination, seedling emergence from the soil and/or plant development,and that also maintain an elevated sugar level, resulting in a sweet orother desirable flavor of the plant, plant material and/or seed over aperiod of time immediately following the prime eating stage thereof.Those having ordinary skill in the art may determine the number of timesthat a particular step in the above process, such as the backcrossing ofnear isogenic lines, should or must be performed in order to achieve asuccessful result of the process. As is indicated above, some of thesesteps are preferably performed multiple times.

In another aspect, the present invention provides a hybrid plant, plant,plant material or seed that has an enhanced vigor in comparison with aconventional mutant shrunken-2 (sh-2) or shrunken-2i (sh2-i) hybridplant, plant, plant material or seed, and an enhanced ability to retainsugar over a period of time immediately following a prime eating stagethereof, in comparison with a conventional mutant sugary-1 (su1) orshrunken-2i (sh2-i) hybrid plant, plant, plant material or seed,consisting of steps (a) through (h) above in any suitable order.

In yet another aspect, the present invention provides a plant seed thatis produced by any one of the methods that is described above (orotherwise herein).

In another aspect, the present invention provides a plant or plantmaterial that is produced by any one of the methods that is describedabove (or otherwise herein).

In still another aspect, the present invention provides a method forproducing a hybrid plant, plant material, or seed comprising crossing aninbred plant line that is homozygous recessive for the mutantshrunken2-i endosperm allele, and also homozygous recessive for one ormore other endosperm alleles, as the female parent plant line with adifferent male parent plant line, which may be, for example, aconventional male parent sweet corn or other plant line.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph that provides a generalized comparison ofrepresentative endosperm sucrose levels for the conventional sugary-1(su1), sugary enhancer-1 (se1) and shrunken-2 (sh2) genetic mutant linesat the prime eating stage (at a level of approximately 75% moisture).

FIG. 2 is a line graph that shows the relative endosperm sugar retentionor “holding ability” at room temperature for the conventional sugary-1(su1), sugary enhancer-1 (se1) and shrunken-2 (sh2) genetic mutant linesat the prime eating stage (at a level of approximately 75% moisture)through a seven day interval (Days 1-7). FIG. 2 shows representativechanges in endosperm sucrose levels over time for the three differentgenetic types.

FIG. 3 is a bar graph that provides a comparison of representativepericarp levels for the conventional sugary-1 (su1), sugary enhancer-1(se1) and shrunken-2 (sh2) genetic mutant lines at the prime eatingstage (at a level of approximately 75% moisture).

FIG. 4 is a line graph that shows the relative pericarp levels at roomtemperature for the conventional sugary-1 (su1), sugary enhancer-1 (se1)and shrunken-2 (sh2) genetic mutant lines at the prime eating stage (ata level of approximately 75% moisture) through a seven day interval(Days 1-7). FIG. 4 shows representative changes in pericarp levels overtime for the three different genetic types.

FIG. 5 is a diagram of the wildtype sh2 gene (sh2-R), which is composedof 16 exons (represented by boxes in FIG. 5) that are separated byintrons (represented by lines in FIG. 5). This gene is approximately6,000 base pairs long. The sh2-R gene has a large (at least 7,800 basepair) insertion in the intron 2-exon 4 region (represented by thetriangle in FIG. 5). Primers (represented by the arrows in FIG. 5)bordering this insertion will not yield a Polymerase Chain Reaction(PCR) product because of the large size of the insertion. In contrast,the sh2-i gene has a single base pair change at the end of intron 2, andprimers flanking the insertion site of sh2-i will yield a PCR product.

FIG. 6 is a line graph that shows mean starch organoleptic scores(organoleptic averages for starch accumulation) for conventional hybridshrunken-2 (sh2) near isogenic lines (NILS), either containing, or notcontaining, the sh2-i allele over time in days 1-7 past the prime eatingstage (at a level of approximately 75% moisture).

FIG. 7 is a line graph that shows organoleptic average sugar (sweetness)scores for the sweet corn varieties Beyond (not including the sh2-igene), Passion (not including the sh2-i gene) and ACX SS 7501Y(including the sh2-i gene) in the seven day period immediately followingthe prime eating stage (at a level of approximately 75% moisture). Itillustrates the comparative organoleptic sugar levels among these threesweet corn varieties over this seven-day period of time.

FIG. 8 is a line graph that shows organoleptic average pericarptenderness scores for the sweet corn varieties Beyond (not including thesh2-i gene), Passion (not including the sh2-i gene) and ACX SS 7501Y(including the sh2-i gene) in the seven day period immediately followingthe prime eating stage (at a level of approximately 75% moisture). Itillustrates the comparative organoleptic pericarp tenderness levelsamong these three sweet corn varieties over this seven-day period.

FIG. 9 shows a schematic representation of the genomic sequence bearingthe splice site alterations of the mutant shrunken-2i (sh2-i) allele(exon 1-4). The point mutation that altered the 3′ splice site AG to AAof intron 2 in mutant sh2-i is boxed. Arrows joined by lines mark thedonor and acceptor sites used during RNA splicing to generate the mutanttranscripts.

FIG. 10 (FIGS. 10A-10U) is a molecular map of samples of individualinbred NILs that were prepared in the experiments that are described inExample 1.

FIG. 11 is a line graph that shows organoleptic average sugar(sweetness) scores for the sweet corn inbred parent lines AC 157Y (notincluding an sh2-i gene) and AC 157Y-i (an sh2-i conversion inbredincluding an sh2-i gene) in the seven day period immediately followingthe prime eating stage (at a level of approximately 75% moisture). Itillustrates the comparative organoleptic sugar levels among these twosweet corn inbred parent lines over this seven-day period of time.

FIG. 12 is a line graph that shows organoleptic average pericarptenderness scores for the sweet corn inbred parent lines AC 157Y (notincluding an sh2-i gene) and AC 157Y-i (an sh2-i conversion inbredincluding an sh2-i gene) in the seven day period immediately followingthe prime eating stage (at a level of approximately 75% moisture). Itillustrates the comparative organoleptic pericarp tenderness levelsamong these two sweet corn inbred parent lines over this seven-dayperiod of time.

FIG. 13 is a line graph that shows organoleptic average sugar(sweetness) scores for the sweet corn varieties Beyond (not includingthe sh2-i gene), Passion (not including the sh2-i gene) and hybrid ACXSS 1082Y sh2-i (an sh2-i conversion inbred including the sh2-i gene) inthe seven day period immediately following the prime eating stage (at alevel of approximately 75% moisture). It illustrates the comparativeorganoleptic sugar levels among these three sweet corn varieties overthis seven-day period of time.

FIG. 14 is a line graph that shows organoleptic average pericarptenderness scores for the sweet corn varieties Beyond (not including thesh2-i gene), Passion (not including the sh2-i gene) and hybrid ACX SS1082Y sh2-i (an sh2-i conversion inbred including the sh2-i gene) in theseven day period immediately following the prime eating stage (at alevel of approximately 75% moisture). It illustrates the comparativeorganoleptic pericarp tenderness levels among these three sweet cornvarieties over this seven-day period.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO. 1 is the 7739 base pair genomic nucleotide sequence of a wildtype Shrunken-2 (sh2) allele of Zea Mays, which is also published inU.S. Pat. No. 6,184,438 B1. (FIG. 1 of U.S. Pat. No. 6,184,438 B1, whichis incorporated herein in its entirety by reference, also shows thegenomic nucleotide sequence of a wild type Shrunken-2 (sh2) allele ofZea Mays. Introns are indicated by lower case letters. Base number 1 isthe transcription start site. The arrow indicates the 3′-end of cDNA.Putative TATA, RY dyad and enhancer sequences are underlined.)

SEQ ID NO. 2 is the cDNA sequence encoding the sugary-1 (su1) allele ofZea Mays, which is also published in U.S. Pat. No. 5,912,413. The 2712base pair nucleotide sequence of the su1 cDNA clone includes a sequenceof 14 consecutive T residues located at one end of the clone,identifying the polyadenylation site and the 3′ end of the mRNA. Acontinuous open reading frame (ORF) of 789 codons begins 88 nucleotidesfrom the 5′ end of the cDNA clone and terminates 240 nucleotides priorto the poly(A) adenylation site. This ORF corresponds to a polypeptideof 789 amino acids (SEQ ID NO. 3). Comparison of the cDNA and a partialgenomic sequence (SEQ ID NO: 4) identifies four exons and introns in thegenomic DNA. The four exons extend from nucleotide 658 to nucleotide1107, nucleotide 1352 to nucleotide 1536, nucleotide 1657 to nucleotide1753, and nucleotide 2076 to nucleotide 2227. The exon sequences or thefull sequence of SEQ ID NO: 4 can be used as probes to obtain the fulllength genomic sequence by methods that are well known by those havingordinary skill in the art.

SEQ ID NO. 3 is an amino acid translation of the sugary-1 (su1)nucleotide sequence of the cDNA clone that is shown in SEQ ID NO. 2,which is also published in U.S. Pat. No. 5,912,413.

SEQ ID NO. 4 is a partial sugary-1 (su1) genomic nucleotide sequence,which is also published in U.S. Pat. No. 5,912,413.

SEQ ID NO. 5 is the deduced amino acid sequence of su1 (SEQ ID NO. 4),which is also published in U.S. Pat. No. 5,912,413.

SEQ ID NO. 6 is a nucleotide sequence of the primer umc1551 (a primerfor molecular markers for the sugary enhancer-1 (se1) allele onchromosome 2).

SEQ ID NO. 7 is a nucleotide sequence of the primer umc1551 (a primerfor molecular markers for the sugary enhancer-1 (se1) allele onchromosome 2).

SEQ ID NO. 8 is a nucleotide sequence of the primer bnlg1520 (a primerfor molecular markers for the sugary enhancer-1 (se1) allele onchromosome 2).

SEQ ID NO. 9 is a nucleotide sequence of the primer bnlg1520 (a primerfor molecular markers for the sugary enhancer-1 (se1) allele onchromosome 2).

SEQ ID NO. 10 is a nucleotide sequence of the primer phi427434 (a primerfor molecular markers for the sugary enhancer-1 (se1) allele onchromosome 2).

SEQ ID NO. 11 is a nucleotide sequence of the primer phi427434 (a primerfor molecular markers for the sugary enhancer-1 (se1) allele onchromosome 2).

SEQ ID NO. 12 is a nucleotide sequence of the primer umc2077 (a primerfor molecular markers for the sugary enhancer-1 (se1) allele onchromosome 2).

SEQ ID NO. 13 is a nucleotide sequence of the primer umc2077 (a primerfor molecular markers for the sugary enhancer-1 (se1) allele onchromosome 2).

SEQ ID NO. 14 is a nucleotide sequence of the primer umc2174 (a primerfor molecular markers for the shrunken-2 (sh2) allele on chromosome 3).

SEQ ID NO. 15 is a nucleotide sequence of the primer umc2174 (a primerfor molecular markers for the shrunken-2 (sh2) allele on chromosome 3).

SEQ ID NO. 16 is a nucleotide sequence of the primer dupssr33 (a primerfor molecular markers for the shrunken-2 (sh2) allele on chromosome 3).

SEQ ID NO. 17 is a nucleotide sequence of the primer dupssr33 (a primerfor molecular markers for the shrunken-2 (sh2) allele on chromosome 3).

SEQ ID NO. 18 is a nucleotide sequence of the primer bmc1257 (a primerfor molecular markers for the shrunken-2 (sh2) allele on chromosome 3).

SEQ ID NO. 19 is a nucleotide sequence of the primer bmc1257 (a primerfor molecular markers for the shrunken-2 (sh2) allele on chromosome 3).

SEQ ID NO. 20 is a nucleotide sequence of the primer umc2277 (a primerfor molecular markers for the shrunken-2 (sh2) allele on chromosome 3).

SEQ ID NO. 21 is a nucleotide sequence of the primer umc2277 (a primerfor molecular markers for the shrunken-2 (sh2) allele on chromosome 3).

SEQ ID NO. 22 is a nucleotide sequence of the primer phi295450 (a primerfor molecular markers for the sugary-1 (su1) allele on chromosome 4).

SEQ ID NO. 23 is a nucleotide sequence of the primer phi295450 (a primerfor molecular markers for the sugary-1 (su1) allele on chromosome 4).

SEQ ID NO. 24 is a nucleotide sequence of the primer phi308090 (a primerfor molecular markers for the sugary-1 (su1) allele on chromosome 4).

SEQ ID NO. 25 is a nucleotide sequence of the primer phi308090 (a primerfor molecular markers for the sugary-1 (su1) allele on chromosome 4).

SEQ ID NO. 26 is a nucleotide sequence of the primer phi076 (a primerfor molecular markers for the sugary-1 (su1) allele on chromosome 4).

SEQ ID NO. 27 is a nucleotide sequence of the primer phi076 (a primerfor molecular markers for the sugary-1 (su1) allele on chromosome 4).

SEQ ID NO. 28 is a nucleotide sequence of the primer phi079 (a primerfor molecular markers for the sugary-1 (su1) allele on chromosome 4).

SEQ ID NO. 29 is a nucleotide sequence of the primer phi079 (a primerfor molecular markers for the sugary-1 (su1) allele on chromosome 4).

The sequences that are present in SEQ ID NOS. 1-29 use abbreviationsthat are described in the World Intellectual Property Organization(WIPO) Handbook on Industrial Property Information and Documentation,Standard ST.25, which is hereby incorporated herein by reference in itsentirety.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention, and to the examples included therein.

DEFINITIONS

For purposes of clarity, various terms and phrases that are usedthroughout this specification and the appended claims are defined in themanner that is set forth below. If a term or phrase that is used in thisspecification, or in the appended claims, is not defined below, orotherwise in this specification, the term or phrase should be given itsordinary meaning.

The term “agronomy” as is used herein means the science of cropproduction.

The term “allele” as is used herein refers to one of multiplealternative form of a gene (one member of a pair) that is located at aspecific position or locus on a specific chromosome, and controls thesame phenotype (with potentially differing effects). Alleles arevariants of a gene that produce different traits in a gene'scharacteristics, and can differ in either coding sequences or non-codingsequences.

The phrase “amino acid” as is used herein means a molecule thatgenerally contains the basic amino group (NH₂), the acidic carboxylicgroup (COOH), a hydrogen atom (—H) and an organic side group (R)attached to the carbon atom, thus having the basic formula ofNH₂CHRCOOH. Amino acids are the building blocks of proteins in whicheach is coded for by a codon and linked together through peptide bonds.More than 100 amino acids have been found to occur naturally, with eachof them differing in their R group. Twenty of them are involved inmaking up a protein, and are classified as whether they arenon-essential or essential. Non-essential amino acids include alanine,arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine,glycine, proline, serine and tyrosine. Essential amino acids includehistidine, isoleucine, leucine, lysine, methionine, phenylalanine,threonine, tryptophan and valine. The abbreviations for amino acids arewell known by those having ordinary skill in the art.

The phrase “backcross” as is used herein means to cross (a hybrid orother plant) with one of its parents, or with an individual that isgenetically identical or similar to one of its parents. It is a cross ofan F₁ to either parent used to generate it.

The term “breeding” as is used herein means the science and/or art ofmanipulating the heredity of an organism for a specific purpose.

The term “carbohydrates” as is used herein means simple organiccompounds comprising carbon, oxygen and hydrogen, generally with manyhydroxyl groups added (usually one on each carbon atom that is not partof the aldehyde or ketone functional group). The basic carbohydrateunits are monosaccharides, such as glucose, galactose and fructose.Carbohydrates have numerous roles in living things, such as the storageand transport of energy (e.g., starch or glycogen) and structuralcomponents (e.g., cellulose in plants). They serve as energy stores,fuels, and metabolic intermediates. Ribose and deoxyribose sugars formpart of the structural framework of RNA and DNA. Polysaccharides arestructural elements that are present in the cell walls of plants.Carbohydrates are linked to many proteins and lipids, where they playkey roles in mediating interactions between cells and interactionsbetween cells and other elements in the cellular environment.Monosaccharides can be linked together into polysaccharides in a largevariety of ways.

The term “cellular respiration” as is used herein means a series ofmetabolic processes that generally take place within a cell in whichbiochemical energy is harvested from an organic substance, such asglucose, and is stored as energy carriers (ATP) for use inenergy-requiring activities of the cell. It consists of glycolysis,citric acid cycle or Krebs Cycle, and oxidative phosphorylation. Thecell appears to “respire” in a way that it takes in molecular oxygen (asan electron acceptor) and releases carbon dioxide (as an end product)(an aerobic process). Cellular respiration is essential to botheukaryotic and prokaryotic cells because biochemical energy is producedto fuel many metabolic processes, such as biosynthesis, locomotion,transportation of molecules across membranes, and the like. While theentire process generally occurs in the cytoplasm of prokaryotes, ineukaryotes, generally glycolysis occurs in the cytoplasm, whereas theKrebs Cycle and oxidative phosphorylation occur in the mitochondrion.While prokaryotic cells can generally yield a maximum of 38 ATPmolecules, eukaryotic cells can generally yield a maximum of 36 ATPmolecules.

The phrase “coding sequence” as is used herein refers to a portion of agene or an mRNA that codes for a protein.

The term “codon” as is used herein means a set of three adjacentnucleotides (triplets), in mRNA that base-pair with the correspondinganticodon of tRNA molecule that carries a particular amino acid, hencespecifying the type and sequence of amino acids for protein synthesis.For example, GCC (Guanine-Cytocine-Cytocine)→alanine, GUU(Guanine-Uracil-Uracil)→valine, CUA (Cytosine-Uracil-Adenine)→leucineand UCA (Uracil-Cytosine-Adenine)→serine.

The phrases “complimentary DNA” and “cDNA” as are used herein mean asingle stranded DNA molecule that is complimentary to a specific RNAmolecule, and that is synthesized from it. Complimentary DNAs areimportant laboratory tools as DNA probes and for isolating and studyingvarious genes.

The phrase “conventional mutant hybrid plant, plant material, seed, lineor variety” as is used herein in connection with a sugary-1 (su1),sugary enhancer-1 (se1), shrunken-2 (sh2), shrunken-2i (sh2-i) or othermutant allele means that the hybrid plant, plant material, seed, line orvariety is a typical hybrid plant, plant material, seed, line or varietythat generally only includes one mutant allele in its genome thatconfers the traits that are described herein, rather than a sequentialor other combination of two or more mutant alleles, for example, onethat includes the mutant shrunken-2i (sh2-i) allele in its genome, butnot the sugary-1 (su1), sugary enhancer-1 (se1) or shrunken-2 (sh2)mutant alleles, or one that includes the mutant sugary-1 (su1) allele inits genome, but not the sugary enhancer-1 (se1), shrunken-2 (sh2) orshrunken-2i (sh2-i) mutant alleles.

The terms “corn” and “maize” as is used herein means any of numerouscultivated forms of a widely grown, usually tall annual cereal grass(Zea mays) bearing grains or kernels on large ears, and includes thenumerous varieties of sweet corn and supersweet corn. The grains orkernels of this plant may be used as food for humans and livestock, orfor the extraction of an edible oil or starch. The kernels may be eatenraw or cooked, and may be canned, frozen and/or stored in other mannersthat are known by those of ordinary skill in the art.

The terms “cross,” “crossing,” “interbreeding” and “crossbreeding” asare used herein mean the act of mixing different species or varieties ofplants to produce hybrids. A monohybrid cross is a breeding experimentbetween parental generation organisms that differ in one trait. In amonohybrid cross, there is generally a genetic cross between parentsthat differ in the alleles they possess for one particular gene, withone parent having two dominant alleles, and the other parent having tworecessive alleles. All of the offspring (monohybrids) then have onedominant and one recessive allele for that gene (i.e. they are hybrid atthat one locus). Crossing between these offspring yields acharacteristic 3:1 (monohybrid) ratio in the following generation ofdominant:recessive phenotypes. For example, the allele for green podcolor (G) is dominant, and the allele for yellow pod color (g) isrecessive. The cross-pollination between a parental generation green podplant and a parental generation yellow pod plant results in all greenoffspring (i.e., all genotypes are Gg). A dihybrid cross is a breedingexperiment between parental generation organisms that differ in twotraits. In a dihybrid cross, there is generally a genetic cross betweenparents that differ in two characteristics, controlled by genes atdifferent loci. Gregor Mendel performed a dihybrid cross using peaplants and the characteristics of seed color and texture. The parentalplants had either smooth yellow seeds (SSYY) (the dominantcharacteristics) or wrinkled green seeds (ssyy) (the recessivecharacteristics). All of the offspring had smooth yellow seeds, beingheterozygous (SsYy) for the two alleles. Crossing between theseoffspring produced an F₂ generation of plants with smooth yellow, smoothgreen, wrinkled yellow, and wrinkled green seeds in the ratio 9:3:3:1.Mendel used these results as the basis for his Law of IndependentAssortment. A trihybrid cross is a breeding experiment between parentalgeneration organisms that differ in three traits, and so forth.

The term “crop” as is used herein means the periodic, such as annual orseasonal, yield of any plant that is grown in significant quantities tobe harvested as food, as livestock fodder, as fuel or for any othereconomic (or other) purpose. Many types of crops are used for industrialpurposes, for example, they are grown and harvested for the sole purposeof making profit and feeding people, and are grown in large quantitiesin certain areas that are suitable for growing crops.

The term “dominant” as is used herein means an allele or a gene that isexpressed in an organism's phenotype, generally masking the effect ofthe recessive allele or gene, when present. It is a phenotype that isexpressed in an organism whose genotype may be either homozygous orheterozygous for the corresponding allele. In genetics, the dominantallele or gene is the one that determines the phenotype of an organism.Its effects are readily recognized in comparison with the effects of therecessive allele or gene. Usually, a dominant allele is symbolized witha capital letter, and a recessive allele is symbolized with a smallletter, for example: Hh (where H refers to the dominant allele and hrefers to the recessive allele).

The phrase “dry seed stage” as is used herein means the first temporalevent in the germination of a plant, such as sweet corn, and in whichthe seed or kernel contains a moisture content that is generally lessthan about 12%.

The phrases “DNA probe,” “gene probe” and “probe” as are used hereinmean a single-stranded DNA molecule used in laboratory experiments todetect the presence of a complimentary sequence among a mixture ofvarious single-stranded DNA molecules.

The phrase “DNA sequencing” as is used herein means a determination ofthe order of nucleotides in a specific DNA molecule.

The phrases “Duncan's New Multiple Range Test” and “MRT” as are usedherein mean a multiple comparison statistical procedure that uses thestudentized range statistic q_(r) to compare sets of means, and isparticularly protective against false negative (Type II) error. Thistest is commonly used in agronomy and in other types of agriculturalresearch, and is well known by those having ordinary skill in the art.Additional information about this test is present in D. B. Duncan,“Multiple Range and Multiple F Tests,” Biometrics 11:1-42, 1955.Statistical procedures that are employed herein, and relatedcalculations, may be performed on computers in a manner, and usingsoftware, that is known by those having ordinary skill in the art.

The term “embryo” as is used herein means a young plant developed froman ovum sexually or asexually and, in seed plants, contained within theseed.

The term “endosperm” as is used herein means the nutritive tissue thatis found in many seeds of plants, and that surrounds the embryo withinsuch seeds. It supplies nutrients to the embryo. The endosperm isgenerally the site of most starch deposition during kernel developmentin maize, and endosperm starch content comprises approximately 70% ofthe dry weight of the kernel or seed.

The term “energy” as is used herein means the ability to do work orproduce change. Energy exists in different forms, but is neither creatednor destroyed. It simply converts to another form, and can be expressedin joules or ergs. Energy is often stored by cells in biomolecules, suchas carbohydrates (sugars) and lipids. The energy is generally releasedwhen these molecules have been oxidized during cellular respiration, andis carried and transported by ATP, an energy-carrier molecule.

The term “enzyme” as is used herein means a protein (or protein-basedmolecule) that generally speeds up a chemical reaction in a livingorganism, such as a plant. Enzymes generally act as catalysts forspecific chemical reactions, converting a specific set of reactants(substrates) into specific products. Enzyme generally have acharacteristic sequence of amino acids that fold to produce a specificthree-dimensional structure, which gives the molecule unique properties,and are usually classified and named according to the reaction that theycatalyze.

The term “epistasis” as is used herein means that a mutation in one genemasks the expression of a different gene. (In contrast, with dominance,one allele of a gene masks the expression of another allele of the samegene.)

The term “exon” as is used herein refers to those portions of a genomicDNA sequence that will be represented in a final, mature mRNA (i.e., acontiguous segment of genomic DNA that codes for a polypeptide in agene). The term “exon” may also refer to equivalent segments in a finalRNA. Exons may include coding sequences, a 5′ untranslated region and/ora 3′ untranslated region.

The term “express” as is used herein means to manifest the effects of agene, to cause to produce an effect or a phenotype, or to manifest agenetic trait, depending upon the context. The expression of a gene isthe translation of information encoded in the gene into protein or RNA.Expressed genes include genes that are transcribed into messenger RNA(mRNA) and then translated into protein, as well as genes that aretranscribed into types of RNA, such as transfer RNA (tRNA) and ribosomalRNA (rRNA) that are not translated into protein. Several steps in thegene expression process may be modulated, including the transcription,RNA splicing, translation and post-translational modification of aprotein. Gene regulation gives the cell control over structure andfunction, and is the basis for cellular differentiation, morphogenesisand the versatility and adaptability of an organism, such as a plant.

The term “fitness” as is used herein refers to a measure of the relativebreeding success of an organism, such as a plant, or genotype, in agiven population at a given time. Individuals that contribute the mostoffspring to the next generation are the fittest. Fitness thereforereflects how well an organism is adapted to its environment, whichdetermines its survival.

The term “gene” as is used herein refers to the basic unit of heredity(genetic traits) in a living organism (plant, animal or micro-organism)that holds the information that is required to pass genetic traits tooffspring. It is a segment of deoxyribonucleic acid (DNA) thatcontributes to a phenotype/function. The DNA is a molecule in the shapeof a double helix, with each rung of the spiral ladder having two pairedbases selected from adenine (A), thymine (T), cytosine (C) or guanine(G). Certain bases always pair together (AT and GC), and differentsequences of base pairs form coded messages. Genes are arranged inprecise arrays all along the length of chromosomes, which are muchlarger structures.

The phrase “gene expression” as is used herein means the process inwhich a cell produces the protein and, thus, the characteristic, that isspecified by a gene's nucleotide sequence.

The phrase “genetic map” as is used herein means a diagram that showsthe genetic linkage relationships among loci on chromosomes (or linkagegroups) within a given species. “Mapping” is the process of defining thelinkage relationships of loci through the use of genetic markers,populations that are segregating for such markers, and standard geneticprinciples of recombination frequency. A “map location” is a specificlocus on a genetic map where an allele can be found within a givenspecies.

The phrase “genetic marker” as is used herein means a specific fragmentof DNA that can be identified within a whole genome. It is a geneticfactor that can be identified and, thus, act to determine the presenceof genes or traits linked with them, but not easily identified.

The term “genome” as is used herein means the complete set of genes inan organism, such as a plant, or the total genetic content in one set ofchromosomes, depending upon the context. It is the complete set ofchromosomes found in each cell nucleus of an individual or organism.

The term “genotype” as is used herein refers to an organism's inheritedinstructions that it carries within its genetic code. A genotype for agene is generally the set of alleles that it possesses. A genotype isthe specific combination of alleles present at a single locus in thegenome.

The term “germination” as is used herein means the process by which adormant seed emerges from a period of dormancy, and begins to sprout andgrow into a seedling under the right growing conditions. The most commonexample of germination is the sprouting of a seedling from a seed of anangiosperm or gymnosperm. It is the growth of an embryonic plantcontained within a seed, and results in the formation of a seedling.

The term “glucose” as is used herein means a simple monosaccharide sugarthat serves as the main source of energy, and as an important metabolicsubstrate for most living things. Its chemical formula is C₆H₁₂O₆.Glucose is one of the products of photosynthesis in plants, and theglucose molecules are stored as repeating units of sugar (e.g. starch).Glucose also serves as an important metabolic intermediate of cellularrespiration.

The term “glume” as is used herein means a basal, membranous, outersterile husk or bract in the flowers of grasses, sedges and maize.

The term “harvest” as is used herein means the gathering (collectingand/or assembling) of a crop of any kind, for example, of maize.

The term “heterozygous” as is used herein means having dissimilaralleles that code for the same gene or trait. It is a situation in whichthe two alleles at a specific genetic locus are not the same. An exampleis a zygote having one dominant allele and one recessive allele, i.e.,Aa, for a particular trait.

The term “heterozygosity” as is used herein means the presence ofdifferent alleles at one or more loci on homologous chromosomes.

The term “homologous” as is used herein in connection with chromosomesmeans those that contain identical linear sequences of genes, and whichpair during meiosis. It means stretches of DNA that are very similar insequence, so similar that they tend to stick together in hybridizationexperiments. Each homologue is a duplicate of one of the chromosomescontributed by one of the parents, and each pair of homologouschromosomes is normally identical in shape and size. Homologous can alsobe used to indicate related genes in separate organisms controllingsimilar phenotypes.

The phrase “homologous chromosomes” as is used herein means a pair ofchromosomes containing the same linear gene sequences, each derived fromone parent.

The term “homozygous” as is used herein means a situation in which twoalleles at a specific genetic locus are identical to one another.

The term “homozygosity” as is used herein means the presence ofidentical alleles at one or more loci (a specific place on a chromosomewhere a gene is located.) in homologous chromosomal segments.

The term “hybrid” as is used herein means an offspring or progenyresulting from a cross between parents of two different species,sub-species, races, cultivates or breeding lines (i.e., fromcrossbreeding). A single cross hybrid is a first generation of offspringresulting from a cross between pure bred parents. A double cross hybridis offspring resulting from a cross between two hybrids of single cross.A three-way cross hybrid is offspring from a cross between a singlecross hybrid and an inbred line. A triple cross hybrid is offspringresulting from the crossing of two different three-way cross hybrids.

The term “hybridization” as is used herein means the process of pollentransfer from the anther of the flower of one plant to the stigma of theflower of a different plant for the purpose of gene transfer to producean offspring (“hybrid”) having a mixed parental genotype. An example ofa cross of two parental lines to produce a hybrid that is discussed inU.S. Pat. No. 4,630,393, for example, is the following:

The genetic composition of the triploid (3N) endosperm of F2 corn earsproduced from plants grown using the above seed is as follows, and theears are characterized by a 3:1 segregation of normal kernels toshrunken (sh2sh2) kernels:

Genotype Phenotype Ratio su1su1su1 Sh2Sh2Sh2 Sugary 1 su1su1su1Sh2Sh2sh2 Sugary 1 su1su1su1 Sh2sh2sh2 Sugary 1 su1su1su1 sh2sh2sh2Sugary, shrunken 1 (have a higher sugar content)Many other examples of crosses are present in this patent. Dependingupon the context, which may readily be determined by those havingordinary skill in the art, the term “hybridization” may alternativelymean bringing complementary single strands of nucleic acid together sothat they stick and form a double strand. In this manner, hybridizationis used in conjunction with DNA and RNA probes to detect the presence orabsence of specific complementary nucleic acid sequences.

The phrase “Independent Assortment” as is used herein refers to aseparation of the alleles of one gene into the reproductive cells(gametes) independently of the way in which the alleles of other geneshave segregated. By this process, all possible combinations of allelesshould occur equally frequently in the gametes. In practice, this doesnot always happen because alleles that are situated on the samechromosome tend to be inherited together. However, if the allele pairsAa and Bb are on different chromosomes, the combinations AB, Ab, aB, andab will normally be equally likely to occur in the gametes.

The term “inbred” as is used herein means offspring produced byinbreeding (succeeding generations of organisms, such as plants, thatare all derived by breeding from the same group of closely relatedorganisms). When lines are inbred sufficiently, a homozygous conditionof particular alleles can generally be assumed.

The term “inbreeding” as is used herein means the breeding of plants,plant materials or organisms that are related, depending upon thecontext (i.e., of plants, plant materials or organisms within anisolated or a closed group of plants, plant parts or organisms). It isthe continued breeding of closely related plants, plant parts ororganisms, so as to preserve desirable traits in a therein.

The term “intron” as is used herein refers to portions of genomic DNAthat are not coding sequences. While they are transcribed (and thuspresent in the primary transcript), they are later spliced out. They,thus, are not present in the corresponding mature mRNA.

The term “isogenic” as is used herein means having the same genotype(i.e., genetically uniform), as all organisms produced by an inbredstrain.

The terms “library,” “DNA library” and “gene library” as are used hereinrefer to a plurality or collection of DNA fragments of one or moreorganisms, each generally carried by a plasmid or virus and cloned intoan appropriate host. A DNA probe is generally used to locate a specificDNA sequence in the library. A collection representing the entire genomeof an organism is known as a genomic library, and an assortment of DNAcopies of messenger RNA produced by a cell is known as a complimentaryDNA (cDNA) library.

A “linkage map” as is used herein means a map of the relative positionsof genetic loci on a chromosome, determined on the basis of how oftenthe loci are inherited together. Distance may be measured incentimorgans (cM).

The term “locus” as is used herein refers to a specific chromosomelocation in the genome of a species where a specific type of gene can befound. It is the position on the chromosome where the gene for aparticular trait resides. A locus may be occupied by any one of severalalleles (variants) for a given gene.

The phrase “Mendel's Laws” as is used herein refers to the two laws thatsummarize Gregor Mendel's theory of inheritance, which are thefoundation of genetics. The Law of Segregation states that eachhereditary characteristic is controlled by two ‘factors’ (alleles),which segregate (separate) and pass into separate germ (reproductive)cells. The Law of Independent Assortment states that pairs of ‘factors’(alleles) segregate independently of each other when germ cells areformed.

The phrase “molecular marker” as is used herein means a specificfragment of DNA that can be identified within a whole genome. It is anidentifiable physical location on a chromosome (i.e., restriction enzymecutting site, gene, or the like) whose inheritance can be monitored.Molecular markers are generally found at specific locations of a genome,and are used to ‘flag’ the position of a particular gene or theinheritance of a particular characteristic. In a genetic cross, thegenes producing characteristics of interest will usually stay linkedwith the molecular markers in relatively close proximity on thechromosome. Thus, varieties can be selected in which the molecularmarker is present, since the marker indicates the presence of thedesired characteristic. Examples of molecular markers include simplesequence repeats (SSRs), single nucleotide polymorphisms (SNPs),randomly amplified polymorphic DNA (RAPDs), and restriction fragmentlength polymorphisms (RFLPs). Additional information about the use ofmolecular markers for use in characterizing and identifying maize inbredlines, validating pedigree and showing associations among inbred linesis present in J. S. Smith et al., “An Evaluation of the Utility of SSRloci as Molecular Markers in Maize (Zea Mays L.): Comparisons with Datafrom RFLPS and Pedigree,” Theor Appl Genet 95:163-173 (1997).Microsatellites, or simple sequence repeats (SSRs) are relatively shortnucleotide sequences, usually from 2 to 3 bases in length that aregenerally repeated in tandem arrays. Amplifiable polymorphisms arerevealed because of differences in the number of tandem repeats that liebetween sequences that are otherwise conserved for each locus.Microsatellite loci are highly polymorphic and are useful as geneticmarkers in many plant species, including maize.

The terms “multiple,” “number” and “plurality” as are used herein meanmore than one, for example, two, three, four, five, six, seven, eight,nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,seventeen, eighteen, nineteen, twenty, twenty five, thirty, thirty-five,forty, forty-five, fifty, fifty-five, sixty and so forth. The term“number” may also include one.

The term “mutation” as is used herein refers to a permanent, heritablechange of genetic material, either in a single gene or in the numbers orstructures of the chromosomes. A mutation occurs when a gene is changedin such a way as to alter the genetic message carried by that gene. Oncethe gene has been changed, the mRNA transcribed from that gene will nowcarry an altered message, and the polypeptide made by translating thealtered mRNA will now contain a different sequence of amino acids. Thefunction of the protein made by folding this polypeptide may also bechanged or lost. In subtle or very obvious ways, the phenotype of theorganism carrying the mutation may be changed. Mutation may be smallscale (affecting the nucleotide sequence of a gene) or large scale(involving a change in the chromosome). It may arise from deletions (thedeletion of one or more nucleotides in the genetic material), insertions(an insertion of one or more extra nucleotides into a new place in thegenetic material) or substitutions (an exchange of one or morenucleotides for another in the genetic material, for example, switchingan A to a G), as may be caused by exposure to ultraviolet or ionizingradiation, chemical mutagens, viruses or the like. For example, asubstitution may: (i) change a codon to one that encodes a differentamino acid, and cause a small change in the protein produced; (ii)change a codon to one that encodes the same amino acid, and causes nochange in the protein produced; or (iii) change an amino-acid-codingcodon to a single “stop” codon and cause an incomplete protein.Mutations may result in the creation of a new character or trait.Mutations may increase an organism's fitness, which may spread throughthe population over successive generations by natural selection.Mutation is the ultimate source of genetic variation, and a particularmutant gene or allele may be compared to its corresponding wild typegene or allele to determine the differences between the two genes oralleles. There are different types of mutations. For example, a pointmutation is a single nucleotide substitution within a gene, and theremay be several point mutations within a single gene. Point mutationsgenerally do not lead to a shift in reading frames and, thus, at mostgenerally cause only a single amino acid substitution. Becauseprotein-coding DNA is divided into codons that are three bases long,insertions and deletions can alter a gene so that its message is nolonger correctly parsed. These changes are known as “frameshifts.” Inframeshifts, a similar error occurs at the DNA level, causing the codonsto be parsed incorrectly. This usually generates truncated proteins thatuseless. Additional information regarding mutations is present inMutation: Science of Everyday Things (Gale Group, 2002).

The term “NILs” as is used herein means near isogenic lines, which arelines of a plant, such as sweet corn, that are genetically identical,except for one locus or a few loci.

The phrase “organoleptic testing” as is used herein means a testing ofthe physical and/or chemical changes that are inherent to decomposition.It may be performed on food, such as sweet corn, to measure and evaluatethe temperature, taste, smell, texture and/or other properties that arecapable of eliciting a response in the sensory organs of human beings oranimals. Organoleptic refers to the sensory properties of a substance,such as taste, color, odor and/or feel, and organoleptic testinginvolves inspection through tasting, feeling, smelling and/or visualexamination of a substance.

The term “phenotype” as is used herein means an observablecharacteristic or trait of an organism, such as sweet corn, such as itsmorphology, development and/or biochemical or physiological properties.It is a biological trait or characteristic possessed by an organism(including a plant) that results from the expression of a specific gene.Phenotypes generally result from the expression of an organism's genes,as well as the influence of environmental factors, and possibleinteractions between the two. In natural populations, most phenotypicvariation is continuous, and is effected by alleles at one or multiplegene loci.

The term “plant” as is used herein means any organism that that belongsto Kingdom Plantae, and that is often characterized by one or more (orall) of the following features:

-   -   an ability to make its own food by photosynthesis (i.e. capable        of capturing energy via the green pigment (chlorophyll) inside        of the chloroplast, and of using carbon dioxide and water to        produce sugars as food and oxygen sugars as food, and oxygen as        byproduct;    -   foods are stored in forms of sugars and starch;    -   a presence of rigid cell walls apart from the cell membrane;    -   has eukaryotic cells (i.e. the presence of a distinct nucleus        surrounded by a membrane);    -   mostly are multicellular (i.e. made up of many cells that are        organized to perform a specific function as a unit);    -   unlimited growth at meristems (when present);    -   organs are specialized for anchorage, support and photosynthesis        (e.g. roots, stems, leaves, etc.);    -   response to stimuli is rather slow due to the absence of sensory        organs and nervous systems;    -   limited movements due to a lack of organs for mobility; and/or    -   has a life cycle that involves both sporophytic and gametophytic        phases.        Plants are the major producers in an ecosystem, and they        include, for example, trees, herbs, bushes, grasses, vines,        ferns and mosses. Examples of particular plants include, but are        not limited to, lettuce, tobacco, cotton, corn, rice, wheat,        carrot, cucumber, leek, pea, melon, potato, tomato, sorghum,        rye, oat, sugarcane, peanut, flax, bean, sugar beets, soya and        sunflower plants.

The term “plasmid” as is used herein means any of several generallypigmented cytoplasmic organelles that are found in plant cells, havingvarious physiological functions, such as the synthesis and storage offood.

The term “pleotropic” as is used herein means producing multiple effectsfrom a single gene. For example, in humans, the Marfan gene ispleotropic and can cause long fingers and toes, dislocation of the lensof the eye and dissecting aneurysm of the aorta.

The term “pollen” as is used herein means the fine powder-like materialconsisting of pollen grains that contain the male reproductive cells ofmost plants. Pollen is generally produced by the anthers of seed plants.

The term “pollination” as is used herein means the process by whichplant pollen is transferred, generally from the anther to the stigma(from male reproductive organs to the female reproductive organs) of aplant flower to produce offspring (to form seeds). In flowering plants,pollen is transferred from the anther to the stigma, often by the windor by insects. In cone-bearing plants, male cones release pollen that isusually borne by the wind to the ovules of female cones. The pollengrain generally contains two cells: a generative cell and a tube cell.The generative nucleus generally divides to form two sperm nuclei. Thetube cell generally grows down into the pistil until it reaches one ofthe ovules contained in the ovary. The two sperm generally travel downthe tube and enter the ovule, where one sperm nucleus generally uniteswith the egg. The other sperm nucleus generally combines with the polarnuclei that exist in the ovule, completing a process known as “doublefertilization.” These fertilized nuclei then generally develop into theendocarp, the tissue that feeds the embryo. The ovule itself generallydevelops into a seed that is contained in the flower's ovary (whichripens into a fruit). In gymnosperms, the ovule is exposed (notcontained in an ovary), and the pollen produced by the male reproductivestructures lands directly on the ovule in the female reproductivestructures.

The phrases “polymerase chain reaction” and “PCR” as are used hereinrefer to a technique that is well known by those having ordinary skillin the art for replicating a specific piece of DNA in vitro, even in thepresence of excess non-specific DNA. Primers are added (which initiatethe copying of each strand) along with nucleotides and heat stable Taqpolymerase. By cycling the temperature, the target DNA is repetitivelydenatured and copied. Because the newly synthesized DNA strands cansubsequently serve as additional templates for the same primersequences, successive rounds of primer annealing, strand elongation, anddissociation produce rapid and highly specific amplification of thedesired sequence. PCR also can be used to detect the existence of thedefined sequence in a DNA sample. A single copy of the target DNA, evenif mixed in with other undesirable DNA, can be amplified to obtainbillions of replicates. PCR can be used to amplify RNA sequences if theyare first converted to DNA via reverse transcriptase. PCR buffers,primers, probes, controls, markers, amplification kits, sDNA synthesiskits, general PCR kits, and the like are available from sources that areknown by those having ordinary skill in the art, such as AppliedBiosystems (Foster City, Calif.), and may readily be used by thosehaving ordinary skill in the art in accordance with the presentinvention.

The term “primer” as is used herein means a relatively shortpre-existing polynucleotide chain to which new deoxyribonucleotides canbe added by DNA polymerase.

The phrases “prime eating stage” and “peak eating stage” as are usedherein mean the stage when a plant, such as sweet corn, tastes the mostfavorable or sweetest, which may readily be determined by those havingordinary skill in the art and, for sweet corn, may be when the cornkernels contain approximately 75% moisture. For example, sweet corntends to mature all at once, and when it is past its prime eating stage,the sweetness generally becomes diminished or absent, and is replaced bya bland, starchy flavor, which is not desirable to consumers.

The term “nucleotide” as is used herein means the basic building block(subunits) of nucleic acids, such as DNA and RNA. It is an organiccompound that is generally made up of nitrogenous base, a sugar and aphosphate group. DNA molecule consists of nucleotides in which the sugarcomponent is deoxyribose, whereas the RNA molecule has nucleotides inwhich the sugar is ribose. The most common nucleotides are divided intopurines and pyrimidines based upon the structure of the nitrogenousbase. In DNA, the purine bases include adenine and guanine, while thepyrimidine bases are thymine and cytosine. RNA includes adenine,guanine, cytosine and uracil instead of thymine. Aside from serving asprecursors of nucleic acids, nucleotides also serve as importantcofactors in cellular signaling and metabolism. These cofactors includeflavin adenine dinucleotide (FAD), flavin mononucleotide, adenosinetriphosphate (ATP) and nicotinamide adenine dinucleotide phosphate(NADP). To form a DNA or RNA molecule, generally thousands ofnucleotides are joined together in a long chain. A DNA oligonucleotideis a short piece of DNA composed of relatively few (oligo-) nucleotidebases.

The term “pericarp” as is used herein means the wall of a plant fruit,such as a corn kernel, which generally is developed from an ovary wall,and contains an outer exocarp, a central mesocarp and an inner endocarp.

The term “phytoglycogen” as is used herein means a plant polysaccharidehaving a structure that is similar to glycogen, and similar properties.For example, the phytoglycogen present in sweet corn is a water solubleglycan having an average unit chain length of 13, reflecting a generallyhigh degree of branching.

The term “polynucleotide” as is used herein means an organic polymermolecule that is composed of nucleotide monomers covalently bonded in achain. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) areexamples of polynucleotides that have a distinct biological function.

The term “polysaccharide” as is used herein means any of a class ofcarbohydrates, such as starch and cellulose, consisting of a number ofmonosaccharides that are joined by glycosidic bonds.

The term “protein” as is used herein refers to a large molecule composedof one or more chains of amino acids in a specific order, which isdetermined by the base sequence of nucleotides in the gene that iscoding for the protein. Proteins are required, for the structure,function, and regulation of cells, and each protein has uniquefunctions.

The term “recessive” as is used herein in connection with a gene means agene whose phenotypic effect is expressed in the homozygous state, butis masked in the presence of the dominant allele (i.e. when the organismis heterozygous for that gene). It is a phenotype that is expressed inorganisms (including plants) only if it is homozygous for thecorresponding allele. Usually the dominant gene produces a functionalproduct whereas the recessive allele does not: both 1 dose and 2 dosesper nucleus of the dominant allele, therefore, generally lead to anexpression of its phenotype, whereas the recessive allele is generallyobserved only in the complete absence of the dominant allele.

The letter “R” as used herein in connection with a designation of asweet corn hybrid indicates that such hybrid is resistant to common leafrust (i.e., that it is rust resistant). Common rust, caused by Pucciniasorghi, is known to be a major pathogen to, and can significantly reducethe yields of, Zea mays, sweet corn. Epidemics of this disease can causeserious losses in yield and quality of sweet corn. High rustsusceptibility of many popular sweet corn hybrids is a major factorcontributing to rust epidemics. Another factor is that sweet corn isusually planted over an extended period from May through June for freshand processing uses. The staggered planting schedules result in highconcentrations of fungal spores in the air, originating from earlyplanted fields, at the time when late-planted fields contain youngactively growing susceptible plants. Common rust on sweet corn appearsin the field as oval to elongate cinnamon brown pustules scattered overupper and lower surfaces of the leaves. The pustules rupture and exposedusty red spores (urediniospores), which are spread by wind and have theability to infect other corn leaves directly. As the pustules mature,they turn brownish black and release the dark-brown overwintering spores(teliospores). In severe epidemics, pustules may also appear on the earsand tassels, and the leaves may yellow and become easily tattered instrong winds. Partial resistance is expressed as chlorotic or necrotichypersensitive flecks with little or no sporulation. Rust epidemics onsweet corn have been severe in the past. Three major factors interact toinfluence the outbreak of rust epidemics on sweet corn: (1) the quantityof urediniospores available to initiate rust epidemics; (2)environmental factors; and (3) the level of rust susceptibility in thesweet corn varieties in use. Urediniospores are unable to overwintersuccessfully in northern climates. Each spring urediniospores move northfrom the southwestern United States and Mexico, following the sequentialplantings of corn from the south up to Canada. Temperatures of 60° to75° F. (16-24° C.) and heavy dews or high relative humidity (close to100%) favor rust development. The current weather conditions influencespore germination and the rate at which rust epidemics develop. Moistureis required for spore germination. Infection will generally occur whenleaves are wet for a minimum of about 3 to about 6 hours. Although mostof the current popular sweet corn hybrids are susceptible to rust,resistant varieties are becoming available. Two types of resistance arebeing used by commercial sweet corn breeders: race-specific resistanceand partial rust resistance. Modest control of rust on sweet corn can beachieved with applications of fungicides. Trials conducted in westernNew York have shown that three applications of mancozeb applied by airsignificantly reduced disease severity on all the leaves of sweet cornplants. Fungicide applications also significantly increased the numberof harvestable ears and the weight of the harvested ears. Researchconducted in other states has shown that, by controlling rust withfungicides, improvements in moisture content sugar content, and ear-tipfill were observed. However, timing of the first fungicide applicationis critical because it needs to be applied early enough to reduce therate of epidemic development. Because rust spores generally arrive fromoutside the immediate area planted to corn, it is often difficult topredict when this spray should be applied.

The letter “SS” as is used herein in connection with a designation of asweet corn hybrid indicates that such hybrid includes the mutant sh2-igene.

The term “seed” as is used herein means a propagating organ formed inthe sexual reproductive cycle of gymnosperms and angiosperms (male andfemale sex cells) that includes a protective coat enclosing an embryoand food reserves. It is a small hard fruit that is generally located ina fertilized ovule of a plant. A seed has two main components, theembryo and the endosperm. The endosperm acts as a food store for theembryo which, over time, will grow from this rich food supply thatenables it to do so. Most seeds go through a period of quiescence inwhich there is no active growth. During this time, the seed can besafely transported to a new location and/or survive adverse climateconditions until it is favorable for growth. The seed contains an embryoand, in most plants, stored food reserves wrapped in a seed coat. Underfavorable growth conditions, a seed begins to germinate, and theembryonic tissues resume growth, developing towards a seedling.Additional information about seeds, seedlings, germination and plantgrowth is present in P. Raven et al., Biology of Plants (7th Edition,New York: W.H. Freeman and Company), 504-508 (2005); and B. Larkins etal., Cellular and Molecular Biology of Plant Seed Development (KluwerAcademic Publishers, 1997).

The term “seedling” as is used herein means a young plant sporophytedeveloping out of a plant embryo from a seed. Seedling developmentstarts with the germination of the seed. A typical young seedlingconsists of three main parts: (i) the radicle (embryonic root); (ii) thehypocotyl (embryonic shoot); and (iii) the cotyledons (seed leaves).During germination, the young plant emerges from its protective seedcoat generally with its radicle first, followed by the cotyledons. Theradicle orients towards gravity, while the hypocotyl orients away fromgravity and elongates through cell expansion to push the cotyledons outof the ground. Typically, seedling development starts withskotomorphogenesis while the seedling is growing through the soil andattempting to reach the light as fast as possible. Generally during thisphase, the cotyledons are tightly closed and form an apical hook toprotect the shoot apical meristem from damage while pushing through thesoil. In many plants, the seed coat still covers the cotyledons forextra protection. Upon breaking the surface and reaching the light, theseedling's developmental program is generally switched tophotomorphogenesis. The cotyledons generally open upon contact withlight (splitting the seed coat open, if still present) and become green,forming the first photosynthetic organs of the young plant. Until thisstage, the seedling generally lives off of the energy reserves that arestored in the seed. The opening of the cotyledons generally exposes theshoot apical meristem and the plumule, consisting of the first trueleaves of the young plant. Seedlings senselight through the lightreceptors phytochrome (red and far-red light) and cryptochrome (bluelight). Once a seedling starts to photosynthesize, it generally is nolonger dependent on the seed's energy reserves. Generally, the apicalmeristems start growing and give rise to the root (the organ of theplant that typically lies below the surface of the soil) and shoot (newplant growth, such as stems and leaves). The first “true” leavesgenerally expand, and can often be distinguished from the roundcotyledons through their species-dependent distinct shapes. While theplant is growing and developing additional leaves, the cotyledonseventually senesce and fall off.

The phrase “segregation analysis” as is used herein means a method forconfirming allelism. This may be performed by crossing two lines thatare homozygous but contain different alleles at a locus in question. Onemay then monitor segregation of the alleles in segregating generationsto test for expected Mendelian segregation patterns. Homology of DNAfragments to the same probe and mutual exclusivity among diverse inbredlines is generally a reasonable test of allelism.

The term “selfing” as is used herein means manually pollinating a plantby placing its pollen on its own stigma (self pollination), and it is abreeding strategy that can lead to homozygosity of an allele. If a setof parents are homozygous for the same allele, this allele is simplytransmitted and the progeny will generally be homozygous for this sameallele. On the other hand, if the parents are homozygous, but fordifferent alleles, A and B, then the progeny is a heterozygote A/B if asingle cross is made, but is homozygous A or B, each with an expectationof ½ if the cross is followed by many selfings.

The phrases “sh2-i mutant allele,” “sh2-i allele,” “sh2-i mutant gene”and “sh2-i gene” as are used herein mean mutant alleles or genes thatcontain the same intron splice site point mutation that is described andshown herein, and confer the same trait(s) as are described herein,whether designated as sh2-i or by some other designation, such as bysh2-N2340.

The term “soil” as is used herein means any kind of a medium, or mixtureof mediums, in which a plant seed, such as a maize seed, will typicallyreasonably grow into a plant, such as the unconsolidated mineral ororganic material that is present on the surface of the Earth, whichserves as a natural medium for the growth of land plants. Such mediumsare known by those having ordinary skill in the art.

The term “starch” as is used herein means a polysaccharide carbohydratethat generally includes a large number of glucose monosaccharide unitsthat are joined together by glycosidic bonds, and is found in plantseeds, bulbs and tubers. Starch is generally predominantly present asamylase and amylopectin. Plants use starch as a way to store excessglucose, and as food during mitochondrial oxidative phosphorylation.

The term “sucrose” as is used herein means a disaccharide made up ofglucose and fructose that is present in many plants, and is widely usedas a sweetener or preservative.

The term “sugar” as is used herein means any disaccharides (e.g.sucrose) and monosaccharides (e.g. fructose or glucose). Sugars areessential structural component of living cells, and are a source ofenergy for many organisms, such as plants. Plants use sugars to storeenergy. Sugars are classified based on the number of monosaccharideunits that are present in the molecule. The monosaccharides join to formmore complex sugars, e.g. disaccharides.

The phrase “test cross” as is used herein means the crossing of anorganism, such as a plant, with an unknown genotype, to a homozygousrecessive organism (tester). It is a cross between an individual ofunknown genotype or a heterozygote (or a multiple heterozygote) to ahomozygous recessive individual.

The term “trait” as is used herein refers to a distinguishing quality orcharacteristic, and is generally a distinct variant of a phenotypiccharacter of an organism, such as sweet corn, that may be inheritedand/or environmentally determined, for example, the sugar content ofsweet corn. The goal of plant breeding in general is to produce progenythat exceed their parents in terms of performance for one or moretraits. Such progeny (transgressive segregants) may be identified usingtechniques that are known by those having ordinary skill in the art,such as segregation analyses. In order to observe transgressivesegregation, parents that complement one another in terms of favorablealleles at various loci must generally be selected. Crossing andrecombination can then result in progeny that contain more favorablealleles than either parent.

The term “transcription” as is used herein means the synthesis of RNAunder the direction of DNA. RNA synthesis, or transcription, is theprocess of transcribing DNA nucleotide sequence information into RNAsequence information. Both nucleic acid sequences use complementarylanguage, and the information is simply transcribed, or copied, from onemolecule to the other. DNA sequence is enzymatically copied by RNApolymerase to produce a complementary nucleotide RNA strand (messengerRNA or mRNA) because it carries a genetic message from the DNA to theprotein-synthesizing machinery of the cell. One significant differencebetween RNA and DNA sequence is the presence of U, or uracil in RNAinstead of the T, or thymidine of DNA. In the case of protein-encodingDNA, transcription is the first step that usually leads to theexpression of the genes, by the production of the mRNA intermediate,which is a faithful transcript of the gene's protein-buildinginstruction. The stretch of DNA that is transcribed into an RNA moleculeis the transcription unit. A DNA transcription unit that is translatedinto protein contains sequences that direct and regulate proteinsynthesis in addition to coding the sequence that is translated intoprotein. The regulatory sequence that is before (upstream (−), towardsthe 5′ DNA end) the coding sequence is the 5′ untranslated region, andregulatory sequence that is found following (downstream (+), towards the3′ DNA end) the coding sequence is the 3′ untranslated region. As in DNAreplication, the RNA is synthesized in the 5′→3′ direction. Only one ofthe two DNA strands is transcribed. This strand is the template strandbecause it provides the template for ordering the sequence ofnucleotides in an RNA transcript. The other strand is the coding strandbecause its sequence is the same as the newly created RNA transcript(except for uracil being substituted for thyamine). The DNA templatestrand is read 3′→5′ by RNA polymerase and the new RNA strand issynthesized in the 5′→3′ direction. A polymerase binds to the 3′ end ofa gene (promoter) on the DNA template strand and travels toward the 5′end. Transcription is divided into 5 stages: pre-initiation, initiation,promoter clearance, elongation and termination. Additional informationabout transcription is present in J. Berg J et al., Biochemistry (6thed., San Francisco: W.H. Freeman, 2006); and R. J. Brooker, Genetics:Analysis and Principles (2nd ed., New York: McGraw-Hill, 2005).

The term “translation” as is used herein means the process by whichpolypeptide chains are synthesized, the sequence of amino acids beingdetermined by the sequence of bases in a messenger RNA, which in turn isdetermined by the sequence of bases in the DNA of the gene from which itwas transcribed. Additional information regarding translation is presentin D. V. Lim, Microbiology (3rd ed., Kendal/Hunt, 2003).

The term “vigor” as is used herein means an exertion of force or ameasure of the increase in plant growth and/or foliage volume throughtime after planting (i.e., after the proper setting of seeds into theground for propagation), and/or of some other superior quality relatedto seed, seedling and/or plant strength and/or growth, such as anenhanced germination and/or seedling emergence out of the ground,depending upon the context. A plant line can be called “vigorous” whenthe line grows vitally, healthy, is tolerant to various biotic andabiotic stresses and/or has a high yield, possibly even while undersub-optimal conditions. Vigor can be measured, and compared fordifferent plant varieties (or for particular lines within a particularplant variety), by methods that are known by those having ordinary skillin the art, in terms of percents (from 0% to 100%), or otherwise, andthe higher the growth and yield of a particular plant variety (or line)(in seeds, fruits, vegetables, plants and/or the like), the more vigorthe plant generally has. For example, one sweet corn variety or line mayproduce approximately 10% fewer kernels when compared with another sweetcorn variety or line. One method for determining plant hybrid “yieldvigor” (and other vigor) is described in U.S. Pat. No. 7,084,320 B2.Other methods for determining vigor are known by those having ordinaryskill in the art.

The term “yield” as is used herein refers to plant, plant materialand/or seed productivity, such as the productivity per unit area of aparticular plant product of commercial significance. For example, yieldof soybean is commonly measured in bushels of seed per acre, or metrictons of seed per hectare, per season.

The term “wildtype” as is used herein refers to a native or predominantgenetic constitution before mutations, usually referring to the geneticconstitution normally existing in nature.

The letter “Y” as is used herein in connection with a designation of asweet corn hybrid indicates that such hybrid is yellow in color.

Abbreviations

The table below lists many of the abbreviations that are employed hereinalong with their meanings.

-   -   su1 (sugary-1) allele: a recessive allele on chromosome 4 (a        mutant), which produce a moderate increase in overall sugar        levels, and resulting sweet taste, to corn, with the sweetness        deteriorating rapidly (thus, a short shelf life), and which        gives corn kernels a smooth and creamy texture and appearance        (desirable);    -   Su1 (starchy-1) allele: a dominant allele on chromosome 4, which        is found in field (dent) corn (not a mutant);    -   se1 (sugary enhancer-1) allele: a recessive allele on chromosome        2 (a mutant), which increases total sugar, and provides an        enhanced sweetness, to conventional su1 variety corn kernels        when the genotype is homozygous se1se1);    -   Se1 allele: a dominant allele on chromosome 2 (not a mutant)        that precludes an expression of elevated sweetness and        tenderness;    -   sh2 (shrunken-2) allele: a recessive allele on chromosome 3 (a        mutant) which produces about twice the sugar content of su1 corn        varieties, and an associated much sweeter taste, when the        genotype is homozygous sh2sh2, which has lightweight,        easily-damaged seeds having a “shrunken” appearance due to a        reduction in endosperm weight and starch levels, and an        associated significantly reduced seedling vigor, fitness and/or        health during germination, seedling emergence from soil, and        plant development and growth in comparison with conventional su1        and se1 sweet corn varieties, and which has a sugar retention at        the post prime eating stage that is significantly extended        relative to conventional su1 and se1 mutant sweet corns;    -   Sh2 allele: a dominant allele present in normal sweet corn        plants (i.e., not including the sh2 mutant allele) (not a        mutant) (See U.S. Pat. No. 4,630,393, col. 3);    -   sh2-i allele: a recessive form of the sh2 gene (a mutant) that        produces enhanced germination and growth characteristics to corn        plants in comparison with corn plants expressing the sh2 gene.

Plant Reproduction: Female and Male Plants

The three structures that generally comprise the reproductive system ofa female plant, which is known as the pistil, are the stigma, style andovary. The stigma is typically that part of the female reproductivestructure upon which pollen adheres and germinates, and is generallyterminal in location. The style is the stalk-like part of the femalereproductive structure, and connects the stigma with the ovary, whichgenerally includes one or more ovules.

The two structures that generally comprise the reproductive system of amale plant, which is known as the stamen, are the anther and thefilament. The anther is the pollen containing part of the malereproductive system, and typically includes one or more sacs. Pollenbecomes formed and matures within this part of the male plant. Thefilament is a stalk-like portion of this reproductive system, andtypically includes the anther at its tip.

During pollination and fertilization, pollen from the male reproductivestructure lands on top of the stigma of the female reproductivestructure and, if conditions are conducive, the pollen will germinate.Upon germination, the pollen forms a pollen tube and grows downwardthrough the style of the female plant and, when it reaches the femaleovary, releases the male nuclei into the ovum, with fertilizationoccurring.

The pericarp of corn (a monocot seed) is the fruit wall or seed coat,which is developed from the ovary wall. The endosperm is locatedinterior to the pericarp, and functions to provide a source of reservematerial and energy for the germination process, during which it will bebroken down and used by the developing embryo for growth. The softendosperm is the soft nutritive tissue formed within the embryo sack ofseed plants, and the hard endosperm is the same nutritive tissue formedin the embryo sack, but is of a hard consistency. The embryo is therudimentary plant located within a seed that will develop into aseedling, and then subsequently into a plant.

Additional information regarding female and male plants, and theirreproduction, is described in T. A. Kiesseelbach, The Structure andReproduction of Corn (Cold Spring Harbor Laboratory Press, 1999); and J.E. Bradshaw, Root and Tuber Crops (Handbook of Plant Breeding).

Information about plant genetics and various plant breeding techniquesis described, for example, in Jack Brown et al., An Introduction toPlant Breeding (Blackwall Publishing LTD, 2008, ISBN 978-1-4051-3344-9);and George Acquaah, Principles of Plant Genetics and Breeding (BlackwallPublishing LTD, 2007, ISBN 13-978-1-4051-3646-4), which includes anextensive glossary of plant genetics and plant breeding terms. Detailsregarding sweet corn cultivation are provided in the web sitesweetcorngrowingtips dot com.

General Description and Utility

The present invention provides unique, cost-effective, reliable,efficient and successful methods for developing and producing plants,plant materials and seeds, such as corn kernels, and corn, that receive,and have, multiple very desirable attributes for consumers of theseproducts, as well as for commercial plant growers, and to improvedand/or enhanced plants, plant materials and seeds that are produced inaccordance with these methods. These inventive methods veryadvantageously provide inbred, hybrid and other plants, plant materialsand seeds that have multiple very beneficial and desirablecharacteristics or traits, generally including those that are describedbelow (as well as others), even when subjected to reasonable amounts ofenvironmental or other stresses, such as cooler temperatures, droughtconditions, low nutrients and other poor soil conditions, crowding,disease, insects, animals, pollution and/or the like. While thesebeneficial traits are described below in connection with corn plants,such traits may also be produced in connection with other types ofplants.

-   -   The corn kernels physically are fuller, and have a higher        carbohydrate and water soluble polysaccharides (WSP) content, at        the dry seed stage in comparison with conventional shrunken-2        (sh2) and shrunken-2i (sh2-i) mutant gene corn varieties (and        other corn varieties, such as wildtype corn varieties), which        have greatly reduced carbohydrate and, thus initial and        subsequent energy levels, and/or water soluble polysaccharides        levels. This very advantageously results in a significantly        enhanced initial and subsequent energy level and growth        characteristics for the plants, such as a stronger (and        maximized) vigor and fitness to the corn during seed        germination, seedling emergence from soil, and plant development        in comparison with the shrunken-2 (sh2) and shrunken-2i (sh2-i)        mutant gene corn varieties (and other corn varieties). The        plants get off to a stronger and more uniform emergence because        of their higher starch reserves, which correlates with sturdier        plants, a larger harvest and more plant yield, all of which is        very desirable to plant growers and home gardeners.    -   Surprisingly, the corn kernels compare favorably in eating        quality with conventional sugary-1 (su 1), sugary enhancer-1        (se1) and shrunken-2 (sh2) sweet corn varieties, and even with        sweet corn varieties including all three of these mutant        alleles, and in some cases are better in eating quality than        such sweet corn varieties (and other sweet corn varieties, such        as wildtype corn varieties). The corn kernels contain elevated        total sugar levels (2 to 3 times the sugar levels of many        conventional corn varieties), resulting in a very desirable        sweet flavor and taste of these kernels, in comparison with        conventional sugary-1 (su1) mutant gene corn varieties, and        other corn varieties, which is very desirable to consumers when        eating the corn kernels. (Corn kernel sugar levels can be        quantified using methods that are known by those having ordinary        skill in the art, such as gas chromatography.) The sugar        retention, and associated sweet taste, of the corn kernels at        the post prime eating stage (particularly in days 1-14        immediately following the prime eating stage) is significantly        extended in comparison with conventional sugary-1 (su1) and        shrunken-2i (sh2-i) mutant gene corn varieties (and other corn        varieties), which have a rapid conversion of sugar to starch        during this period of time (resulting in a loss of sugar), and        an associated reduction in sweet taste of the corn kernels, and        a narrow harvest window before sweetness deteriorates very soon        after the prime eating stage. This sugar retention very        advantageously results in a longer sweet taste of the corn        kernels, which is very desirable to consumers, a longer harvest        window of the corn, a longer holding ability of the corn, and a        longer shelf life of the corn before sweetness deteriorates        after the prime eating stage, which very advantageously provides        a much greater flexibility of harvest, and handling conditions,        of the corn for corn growers. The corn kernels have a reduced        starch accumulation at, and to a practical point following, the        prime eating stage (peak eating quality), such as from about        1-14 days immediately following the prime eating stage, in        comparison with conventional sugary-1 (su1) and shrunken-2i        (sh2-i) mutant gene corn varieties (and other corn varieties).

The corn kernels of hybrid maize varieties that are produced inaccordance with the methods of the invention are smooth and attractive,sweet, tender, plump and creamy, and have a high eating quality, all ofwhich is very desirable to consumers worldwide.

Corn breeders, corn producers, corn growers, scientists and others havenot been able to produce a sweet corn that includes each of the above,and very desirable, production and consumer traits (i.e., these combinedtraits). Further, the present inventor spent more than four yearsconducting experiments to attempt to successfully develop and producehybrid varieties of sweet corn that include these very desirablecombined traits, and that include the shrunken-2i (sh2-i) mutant allelealong with one or more other mutant alleles, and were finallysurprisingly and unexpectedly able to accomplish this goal.

The methods of the present invention combine specific mutant allelesthat are present in sweet corn, or other plants, with the shrunken-2i(sh2-i) gene. The mutant alleles, which include, but are not limited to,the sugary-1 (su1), sugary enhancer-1 (se1), and shrunken-2 (sh2) mutantendosperm alleles, when expressed in a sweet corn (or other plant)hybrid in combination with the shrunken-2i (sh2-i) gene, provideenhanced growth characteristics, such as an enhanced seedlinggermination and seedling vigor, to the sweet corn (or other plants), aswell as the other very beneficial characteristics that are describedhereinabove. The methods of the present invention preferably provide aunique, sequential layering of the mutant alleles su1su1, Su1Su1 and/orse1 se1, in combination with the sh2-i mutant endosperm allele, thatpreserves an enhanced seedling germination and vigor along with productholding ability and shelf life.

The methods of the invention involve the use, and identification of,commercial (and other) hybrid, inbred and other plant lines containingthe above mutant alleles, either singly or in combination, and exceedconventional expectations relative to seedling and plant growthcharacteristics. These mutant alleles confer elevated sweetness, andreduced kernel pericarp, differentially in specific combination.

The problem to be solved by the present invention, and its goal, was tomanipulate the regulation of carbohydrate accumulation and pericarptenderness in sweet corns containing the shrunken-2i (sh2-i) gene. Theexamples that are set forth herein describe experiments that wereperformed in order to solve this problem and achieve this goal.

Plant seeds, plant materials and plants that may be produced inaccordance with the methods of the present invention include those thatare capable of having the shrunken-2i (sh2-i) mutant allele, and atleast one other beneficial mutant allele, including, but not limited to,the mutant sugary-1 (su1), sugary extender-1 (se1) and/or shrunken-2(sh2) mutant endosperm alleles, incorporated into their genome, andexpressed, in a manner that produces the beneficial combined grower andconsumer traits that are described herein, which may be determined bythose having ordinary skill in the art.

In a preferred embodiment, the invention involves a unique sequentialcombination or layering of the shrunken-2i mutant (sh2-i) allele withthe mutant sugary (su1), sugary enhancer-1 (se1) and/or shrunken-2 (sh2)alleles in sweet corn. The unique sequential layering of su1su1, Su1Su1and/or se1se1 in combination with the mutant sh2-i allele functions topreserve enhanced seedling germination and vigor along with productholding ability and shelf life, and provide sweet corn (and otherplants) with the other beneficial traits that are described herein.

Nucleotide Sequences of Mutant Genes

The nucleotide sequences of some of the mutant genes that may beemployed in the methods of the present invention are set forth herein.The nucleotide sequences of other mutant genes that may be employed inthe invention, and related or other nucleotide sequences, may be readilyobtained from sources that are known by those having ordinary skill inthe art, such as from the Maize Genetics and Genomics and/or GenBankdatabases.

The Maize Genetics and Genomics database is a community database forbiological information about the crop plant Zea mays, and is funded bythe USDA Agricultural Research Service. The following data types areaccessible through this site: genetic, genomic, sequence, gene product,functional characterization, literature reference, andperson/organization contact information.

The GenBank sequence database is an open access, annotated collection ofall publicly available nucleotide sequences and their proteintranslations. This database is produced at the National Centers forBiotechnology Information, which is a branch of the National Institutesof Health (Bethesda, Md.), and is available on line via the Entrezsearch engine. GenBank and its collaborators receive sequences producedin laboratories throughout the world from more than 100,000 distinctorganisms, and it continues to grow at an exponential rate, doublingevery 18 months. It contains over 65 billion nucleotide bases in morethan 61 million sequences.

Plant Molecular Work and Molecular Markers Standard materials andmethods for plant molecular work are described by R. D. D. Croy, “PlantMolecular Biology Labfax” (jointly published by BIOS ScientificPublications Ltd (UK) and Blackwell Scientific Publications, UK (1993))and by D. R. Duncan et al., “Methods in Molecular Biology, Plant Celland Tissue Culture” (Humana Press, Clifton, N.J. (1990)).

The experiments that led to the methods of the present invention werefacilitated by the use of genomic marker assisted selection.

Molecular markers can help plant breeders relatively quickly andaccurately select critical traits that enhance plants for theagricultural, horticultural, viticultural, and ornamental industries.They can also help quality assurance personnel make appropriatedecisions with respect to hybrid, varietal and inbred purity.

The application of DNA-based markers allows a plant breeder to identifyphysical characteristics at the molecular level, thus lending ascientific hand in creating and replicating plant varieties. Plantbreeding and seed production programs can be enhanced by applyingmolecular markers for trait selection and mapping, or variety and hybridgenotyping. They provide vehicles for locating and comparing lociregulating quantitative traits requires a segregating population ofplants. Each one may be genotyped using molecular markers.

A molecular marker showing polymorphism between the parents of apopulation which is closely-linked to a gene regulating a particulartrait will mainly co-segregate with that gene and the observable trait(i.e., segregate according to the phenotype if the gene has a largeeffect). Thus, if plants are grouped according to expression of thetrait, and extreme groups are tested with that polymorphic marker, thefrequency of the two marker alleles present within each of the two bulksshould deviate significantly from the ratio of 1:1 expected for mostpopulations. As chromosomal locations of many molecular markers have nowbeen determined in many species, the map location of closely-linkedgenes can, therefore, be deduced without having to genotype everyindividual in segregating populations. This can be used with compositepopulations of maize and other crops and plants to locate quantitativetrait loci that are associated with various traits.

Conventional plant breeding, in contrast, is primarily based uponphenotypic selection of desired individuals among segregating progeniesresulting from directed hybridization. In some instances, plants fromsegregating populations can be grouped according to phenotypicexpression of a trait, and tested for differences in allele frequencybetween the population bulks using bulk segregant analysis (BSA) orother methods. The same probes used for making a genetic map, such asisozyme, RFLP, RAPD, and the like, can be used for BSA. However,although strides have been made in crop improvement through phenotypicselections for agronomically desirable traits, considerable difficultiesare often encountered during such process. These difficulties may arisefrom genotype-environmental interactions, epistatic and pleotropiceffects, or a host of other factors. Sweet corn mutant alleleidentification and quantification is particularly encumbered by the lackof phenotypically assisted identification and selection.

Even if an enzymatic basis for a particular mutant gene is not known,and the nucleotide sequence for the gene encoding the enzyme is notknown, and is not present in the Maize Genetics and Genomics or GenBankdatabases, the inheritance of the gene can still be determined by thosehaving ordinary skill in the art by following nearby molecular markerson the chromosome including the gene, as is described in the Examplessection herein in detail.

Primers for the molecular markers that were used in the Examplesappearing herein are publicly available, and may be found in the MaizeGenetics and Genomics database at the Internet site maizegdb dot org.The following are primers for examples of useful molecular markers.

Primers for Molecular Markers for the se1 Allele on Chromosome 2

umc1551: CACCGGAACACCTTCTTACAGTTT CGAAACCTTCTCGTGATGAGC bnlg1520:TCCTCTTGCTCTCCATGTCC ACAGCTGCGTAGCTTCTTCC phi427434: CAACTGACGCTGATGGATGTTGCGGTGTTAAGCAATTCTCC umc2077: CTGGTTCGGATGCAAGTAGTCAGAAACTCACTGAACATGATCCTGGC

Primers for Molecular Markers for the sh2 Allele on Chromosome 3

umc2174: ACATAAATAAAACGTGTGCCGCAG GTACGTACGCAGCCACTTGTCAG dupssr33:GTGCTTGGGACAAAAAGG AGTCCACTCCAGAGGATG bmc1257: CGGACGATCTTATGCAAACAACGGTCTGCGACAGGATATT umc2277: CTCTTCACGCTCAATAAACCCAGTTAACTGCAGAAACGGTGGTCAATA

Primers for Molecular Markers for the su1 Allele on Chromosome 4

phi295450: CCTTTTCATGTTGCTTTCCC GCCCAATCCTTCCTTCCT phi308090:CAGTCTGCCACGAAGCAA CTGTCGGTTTCGGTCTTCTT phi076: TTCTTCCGCGGCTTCAATTTGACCGCATCAGGACCCGCAGAGTC phi079: TGGTGCTCGTTGCCAAATCTACGAGCAGTGGTGGTTTCGAACAGACAA

Commercially available maize marker libraries, specifically, simplesequence repeats (SSR), may be used for trait identifications, and maybe procured from sources that are known by those having ordinary skillin the art. For example, STA Laboratories (Longmont, Colo.) providescommercially available molecular marker and mapping services inconnection with seeds and plant breeding, as well as hybrid purity andvarietals identification using high resolution Isoelectric FocusingElectrophoresis (IEF). This company provides molecular services for anidentification, and incorporation of, specific sweet corn mutantalleles.

In the experiments that led to the methods of the present invention,commercially available maize marker libraries, specifically, simplesequence repeats (SSR), were obtained for trait identifications, andapproximately 330 SSR markers were tested, primarily targeting thesugary-1 (su1), sugary enhancer-1 (se1) and shrunken-2 (sh2) mutantendosperm allele published genomic chromosomal sites.

In conjunction with the SSR marker libraries, proprietary sweet cornNILs (near isogenic lines) were utilized to evaluate the markerefficacies, as well as to generate specific desirable mutant alleles forcombination with the mutant sh2-i gene. Near isogenic lines of specificinterest were:

-   -   (i) Su1Su1 se1se1 sh2sh2; and    -   (ii) su1su1 se1se1 sh2sh2.

Additional information about the use of molecular marker libraries forplants and genetic maps is present in, A. Kalinski, “Molecular Markersin Plant Genome Analysis” (Diane Publishing Co., 1995); and H. Lorz etal., “Molecular Marker Systems in Plant Breeding and Crop Improvement,”(Springer, 2007).

Conditions for Growing and Harvesting Plants

Those having ordinary skill in the art know how to properly andsuccessfully plant, grow and harvest plants, such as sweet corn.Typically, for example, sweet corn is grown in soil having a pH rangingfrom about 6 to about 6.5 in full sun, with a planting depth of about 1inch. Fertilization is typically performed when the sweet corn plantsreach about 12″ in height for tall varieties, and from about 18″ toabout 24″ in height for other varieties. The sweet corn plants aretypically harvested at approximately 64 days after seedlings emerge. Asis known by those having ordinary skill in the art, the foregoingconditions may be varied.

Publications that describe how sweet corn and other plants can beplanted, grown and harvested include B. R. Lerner et al., “Growing SweetCorn,” Department of Horticulture, Purdue University CooperativeExtension Service, Vegetables HO-98-W, 1-3 (2001); J. R. Schultheis,“Sweet Corn Production,” North Carolina Cooperative Extension Service,North Carolina State University, Revised 12/94; D. L. Larson,“Supersweet Sweet Corn: 50 Years in the Making,” Inside Illinois Vol.23, No. 3 (2003), University of Illinois at Urbana-Champaign NewsBureau; and “Sweet Corn,” Oregon State University, Horticulture 233webpage.

STA Laboratories (Longmont, Colo.) performs physical purity and vigoranalyses using Seedling Vigor Imaging System (SVIS), as well as otherseed analyses services. Registered Seed Technologists (RST) ensureuniform testing standards to meet seed labeling regulations in theUnited Stated and abroad. STA Seed Health Laboratories are USDAaccredited through the National Seed Health System (NSHS).

Other Variations

Deletions, additions, and substitutions of the nucleotide sequencesencoding portions or all of the mutant alleles that are described hereinare contemplated as being within the scope of the present invention, solong as substantially the same phenotype and characteristics observedwith the conventional (unaltered) nucleotide sequence is exemplified.

The nucleotide mutations of introns contemplated within the scope of thepresent invention can also be associated with, or used in conjunctionwith, other mutations of the genes encoding plant AGP polypeptide orencoding other proteins or enzymes. These other mutations include, butare not limited to, mutations in the wild-type sequence that conferother agronomically desirable traits, such as heat stability, diseaseresistance, and other desirable characteristics in a plant expressingthese mutant alleles.

A mutation of the terminal nucleotide of intron 2 of the Shrunken-2(sh2) genomic nucleotide sequence is specifically exemplified herein.However, mutations of the terminal nucleotide in other Shrunken-2 (sh2)introns are also within the scope of the invention, as long as theseconfer substantially the same characteristics to a plant expressing theallele as those associated with the mutation at intron 2, i.e.,germination and seedling vigor comparable to or better than plantsexpressing wild-type Shrunken-2 (sh2) allele, but with enhanced food ortaste quality of the vegetable comparable to, or better than, thatassociated with mutants that provide enhanced sweetness, such as theSh2-R allele, over wild-type. Those having ordinary skill in the art,and having the benefit of the teachings that are described herein, canreadily prepare mutations in other introns of the gene, and determinewhether the mutated introns confer the desired characteristics to theplants.

Plants that are contemplated within the scope of the invention include,for example, maize, sweet peas, tomatoes, bananas and any other plant inwhich a high sucrose content of the vegetable or fruit and germination,seedling and plant growth vigor, are desired characteristics. Otherplants that are contemplated within the scope of the invention includethose that are described elsewhere herein. Also contemplated within thescope of the invention is plant material, such as plant tissue, cells orseeds, that contain the polynucleotides that are described herein.

Additional References

The following additional references may be of interest or helpful incarrying out the present invention: T. Maniatis et al., “MolecularCloning: A Laboratory Manual” (2d Edition, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989); D. R. McCarty, “A SimpleMethod for Extraction of RNA from Maize Tissue,” Maize Genet. Coop.Newslett. 60: 61 (1986); A. Gutierrex-Rojas, “PhenotypicCharacterization of Quality Protein Maize Endosperm Modification andAmino Acid Contents in a Segregating Recombinant Inbred Population,”Crop Sci 48, 1714-1722 (2008); L. Hannah et al., “Characterization ofAdenosine Diphosphate Glucose Pyrophosphorylases from Developing MaizeSeeds,” Plant Physiol. 55:297-302 (1975); L. Hannah et al.,“Characterization of ADP-Glucose Pyrophosphorylase from Shrunken-2 andBrittle-2 Mutants of Maize,” Biochemical Genetics 14(7,8):547-560(1976); M. J. Giroux et al., “ADP-Glucose Pyrophosphorylase inShrunken-2 and Brittle-2 Mutants of Maize,” Molecular & General Genetics243(4):400-408 (1994); L. Shailesh et al., “The AG DinucleotideTerminating Introns is Important but not always Required for Pre-mRNASplicing in the Maize Endosperm,” Plant Physiology 120(1):65-72 (1999);M. Clancy et al., “Maize Shrunken-1 Intron and Exon Regions IncreaseGene Expression in Maize Protoplasts,” Plant Science 98:151-161 (1994);J. Callis et al., “Introns Increase Gene Expression in Cultured MaizeCells.” Genes & Development 1:1183-1200 (1987); K. R. Luehrsen et al.,“Intron Creation and Polyadenylation in Maize are Directed by AU-richRNA,” Genes & Development 8:1117-1130 (1994); V. L. Van Santen et al.,“Splicing of Plant Pre-mRNAs in Animal Systems and Vice Versa” Gene56:253-265 (1987); V. Vasil et al., “Increased Gene Expression by theFirst Intron of Maize Shrunken-1 Locus in Grass Species” Plant Physiol.91:1575-1579 (1989); J. Anderson et al., “The Encoded Primary Sequenceof a Rice Seed ADP-glucose Pyrophosphorylase Subunit and Its Homology tothe Bacterial Enzyme,” The Journal of Biological Chemistry264(21):12238-12242 (1989); J. Anderson et al., “MolecularCharacterization of the Gene Encoding a Rice Endosperm-Specific ADPGlucose Pyrophosphorylase Subunit and its Developmental Pattern ofTranscription,” Gene. 97:199-205 (1991); L. Copeland et al.,“Purification of Spinach Leaf ADPglucose Pyrophosphorylase,” PlantPhysiol. 68:996-1001 (1981); M. Morell et al., “Affinity Labeling of theAllosteric Activator Site(s) of Spinach Leaf ADP-glucosePyrophosphorylase,” The Journal of Biological Chemistry 263(2):633-637(1988); B. Muller-Rober et al., “One of two Different ADP-GlucosePyrophosphorylase Genes from Potato Responds Strongly to Elevated Levelsof Sucrose,” Mol. Gen. Genet., 224:136-146 (1990); P. Nakata et al.,“Comparison of the Primary Sequences of Two Potato Tuber ADP-GlucosePyrophosphorylase Subunits,” Plant Molecular Biology 17:1089-1093(1991); T. Okita et al., “The Subunit Structure of Potato TuberADPglucose Pyrophosphorylase,” Plant Physiol. 93:785-790 (1990); M.Olive et al., “Isolation and Nucleotide Sequences of cDNA ClonesEncoding ADP-Glucose Pyrophosphorylase Polypeptides from Wheat Leaf andEndosperm,” Plant Molecular Biology 12:525-538 (1989); B. Keith et al.,“Monocot and Dicot Pre-mRNAs are Processed with Different Efficienciesin Transgenic Tobacco,” EMBO J. 5(10):2419-2425 (1986); and Z.Kiss-Laszlo et al., “Splicing of Cauliflower Mosaic Virus 35S RNA isEssential for Viral Infectivity,” EMJO J. 14(14):3552-3562 (1995).

Sources of Ingredients, Materials and Equipment

All of the ingredients, materials and equipment that are employed in theexamples, and generally employed in the methods of the invention, arecommercially available from sources that are known by those havingordinary skill in the art, such as Abbott and Cobb, Inc. (Trevose, Pa.),the Maize Stock Center (Urbana/Champaign, Ill.), STA Laboratories(Longmont, Colo.), GenBank (Bethesda, Md.), The Maize Genetics andGenomics Database, the American Tissue Culture Collection (ATCC)(Rockville, Md.), Applied Biosystems (Foster City, Calif.), ResponseGenetics, Inc. (Los Angeles, Calif.), Transgenomic (Omaha, Nebr.),DiaPharma Group, Inc. (West Chester, Ohio), Biomol GmbH (Hamburg,Germany), DxS Ltd. (Manchester, UK), Invitrogen (Carlsbad, Calif.),Syngenta Seeds, Inc. (Stanton, Minn.), Rogers (Wilmington, Del.),Monsanto Corporation (St. Louis, Mo.), Garst Seed Company (Slater,Iowa), Holden Foundation Seed (Williamsburg, Iowa), The University ofFlorida (Gainesville, Fla.), Life Technologies (Gaithersburg, Md.),Alpha Innotech Corporation (San Leandro, Calif.), Amersham InternationalPLC (Arlington Heights, Ill.), and Molecular Dynalics (Sunnyvalle,Calif.). For example, Applied Biosystems sells internationally via itsweb site (applied biosystems dot corn) and otherwise a wide variety ofdifferent products and computer software for conducting DNA sequencing,DNA synthesis (by ligation, Capillary Electrophoresis or the like), DNAand RNA modification and labeling, DNA and RNA purification, geneexpression, genotyping, PCR, peptide synthesis, protein sequencing,transcription, translation, various assays, and the like, such asexpression vectors, probes, primers, which may be readily employed bythose having ordinary skill in the art for carrying out the presentinvention.

The following examples describe and illustrate the methods of thepresent invention. These examples are intended to be merely illustrativeof the present invention, and not limiting thereof in either scope orspirit. Those of ordinary skill in the art will readily understand thatvariations of certain of the conditions and/or steps employed in theprocedures described in the examples can be employed. While theseexperiments have been performed using sweet corn kernels and plants, thesame procedures that are described therein may be employed with otherplant seeds and plants, for example, those that are described elsewhereherein.

The tables below summarizes different sweet corn parent NIL lines andsweet corn hybrid varieties, and their various characteristics (referredto in the Examples as the “Characteristics Tables”), that were preparedin the various examples that are set forth below, and that were assignedparticular designations, including various single, double or triplehomozygous recessive allelic combinations of the sugary (su), sugaryenhancer-1 (se) and shrunken-2 (sh2) mutant genes employed in the maleparent lines, with all hybrids including the sh2-i gene in theirgenomes. In these tables, “in.” refers to inches and “No.” refers tonumber.

Seed deposits made with the American Type Culture Collection (ATCC) aredescribed in two separate sections of the “Examples” section of theapplication, with some (relating to Examples 1-5) being discussed at theend of Example 5 and others (relating to Examples 6-8) being discussedat the end of Example 8.

Example Use of Molecular Number Parent or Hybrid Designation AllelicCombination Markers 2 Hybrid ACX SS 7501Y su1su1 se1se1 sh2sh2-i YesParent (Female) AC 199Y su1su1 se1se1 sh2-i sh2-i Yes Parent (Male) AC195Y su1su1 se1se1 sh2sh2 Yes 3 Hybrid ACX SS 7078Y su1su1 se1se1sh2sh2-i Yes Parent (Female) AC 199Y su1su1 se1se1 sh2-i sh2-i YesParent (Male) AC 128Y su1su1 se1se1 sh2sh2 Yes 3 Hybrid ACX SS 7403RYsu1su1 se1se1 sh2sh2-i Yes Parent (Female) AC 199Y su1su1 se1se1 sh2-ish2-i Yes Parent (Male) AC 215Y su1su1 se1se1 sh2sh2 Yes 4 Hybrid AC151Y Su1su1 Se1se1 sh2sh2-i No Parent (Female) AC 103Y Su1Su1 Se1Se1sh2-i sh2-i No Parent (Male) AC 107Y su1su1 se1se1 sh2sh2 No 4 Hybrid AC188W Su1su1 Se1Se1 sh2sh2-i No Parent (Female) AC 106 W Su1Su1 Se1Se1sh2-i sh2-i No Parent (Male) AC 163W su1su1 Se1Se1 sh2sh2 No 7 HybridACX SS 1082Y sh2-i Su1Su1 Se1Se1 sh2sh2-i No Parent (Female) AC 157Y-iSu1Su1 Se1Se1 sh2-i sh2-i No Parent (Male) AC 144Y Su1Su1 Se1Se1 sh2sh2No 8 Hybrid ACR SS 4500Y Su1su1 Se1se1 sh2sh2-i No Parent (Female) AC098Y-i Su1Su1 Se1Se1 sh2-i sh2-i No Parent (Male) AC 233Y su1su1 se1se1sh2sh2 No 8 Hybrid ACR SS 4501Y Su1su1 Se1Se1 sh2sh2-i No Parent(Female) AC 157Y-i Su1Su1 Se1Se1 sh2-i sh2-i No Parent (Male) AC 241Ysu1su1 Se1Se1 sh2sh2 No

Characteristics Table (Parental Sweet Corn Lines) AC 199Y AC 128Y AC215Y AC 098Y-i AC 233Y AC 157Y-i AC 241Y AC 195Y AC 144Y Maturity  75Day  72 Day  72 Day  79 Day  79 Day  80 Day  76 Day  74 Day  72 DayHeight  66 in.  70 in.  72 in.  60 in.  73 in.  66 in.  63 in.  72 in. 68 in. No. Tillers  1.5  1.8  2.0  0.3  1.6  0.5  1.4  1.8  1.2 GlumeGreen Green Green Green Green Green Green Green Green Color Tassel 14 1010 18 20 18 16 16 16 Laterals Ear 6.8 in. 6.0 in. 6.2 in. 5.8 in. 7.3in. 5.8 in. 6.5 in. 6.8 in. 6.0 in. Length Ear 1.8 in. 1.5 in. 1.5 in.1.8 in. 1.9 in. 1.8 in. 2.0 in.  1.8 1.5 in. Diameter Ear ShapeCylindrical Cylindrical Cylindrical Cylindrical Cylindrical CylindricalCylindrical Cylindrical Cylindrical Kernel  10 mm   6 mm   6 mm   8 mm 12 mm   8 mm  14 mm  10 mm   8 mm Depth No. Kernel 14-16-18 14-1614-16-18 14-16-18 16-18 14-16-18 16-18-20 16-18-20 14-16 Rows KernelYellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Color

Characteristics Table (Hybrid Sweet Corn Lines) ACX SS ACX SS ACX SS ACRSS ACR SS ACX SS 7501Y 7078Y 7403RY 4500Y 4501Y 1082Y Maturity  76 Days  78 Days   75 Days   77 Days  78 Days   79 Days Height  72 in.   80 in.  84 in.   77 in.  73 in.   84 in. No. Tillers  1.5  0.8  1.5  1.5  1.0 1.0 Glume Green Green Green Green Green Green Color Tassel 18 14 12 2428 22 Laterals Ear Length 8.0 in. 8.25 in. 8.25 in. 8.25 in. 8.5 in. 8.5 in. Ear 1.8 in.  2.0 in  1.9 in.  1.8 in. 2.1 in. 1.78 in. DiameterEar Shape Cylindrical Cylindrical Cylindrical Cylindrical CylindricalCylindrical Kernel   9 mm   12 mm   10 mm    9 mm  11 mm   10 mm DepthNo. Kernel 16 16-18 16-18 16-18 16-18-20 16-18 Rows Kernel Yellow YellowYellow Yellow Yellow Yellow Color

Example 1 Construction of Parental Inbred Near Isogenic Lines (NILs)Triple Allelic Combinations of the Sugary (su), Sugary Enhancer-1 (se1)and Shrunken-2 (sh2) Endosperm Mutant Genes

In the experiments that are described in this example, in order toattempt to solve the problem of manipulating the regulation ofcarbohydrate accumulation, and pericarp tenderness, in sweet cornvarieties containing the mutant sh2-i gene, appropriate parental nearisogenic lines (NILs) were constructed and tested, using methods thatare described below and/or known by those having ordinary skill in theart, of the following triple allelic genotype combinations:

-   (1) Su1Su1Se1Se1 sh2sh2=conventional commercial “supersweet” type    with standard eating quality    -   (including one homozygous recessive endosperm mutant allele)-   (2) Su1Su1 se1se1 sh2sh2=conventional commercial “supersweet” type    with very good eating quality    -   (including a double homozygous recessive endosperm mutant        allelic combination)-   (3) su1su1 se1se1 sh2sh2=exotic limited commercial type with    exceptional eating quality    -   (including a triple homozygous recessive endosperm mutant        allelic combination)

Construction of Parental NILs

STA Laboratories (Longmont, Colo.) assisted in providing the molecularservices involved in the identification, and incorporation, of specificsweet corn mutant alleles into the genomes of maize plants.Commercially-available appropriate maize marker libraries, specifically,simple sequence repeats (SSR), were obtained for trait identificationsfrom STA Laboratories, and originated from libraries published byPioneer Hi-Bred International, Inc. (Johnston, Iowa), a developer andsupplier of advanced plant genetics to farmers worldwide. The markerswere all publically available from the maize genetics and genomicswebsite maizegdb dot org. The markers that start with the designation“phi” were developed and released to the public by Pioneer Hi-BredInternational, Inc. Approximately 330 SSR markers were tested, primarilytargeting the su1, se1 and sh2 published genomic chromosomal sites.

The above NILs were backcrossed and self pollinated a sufficient numberof times in a manner known by those of ordinary skill in the art toeffect an adequate reconstitution of the recurrent parents.

Molecular markers were utilized in a manner known by those of ordinaryskill in the art to assist in the identification of su1 and se1 mutantalleles, in particular. These molecular markers were helpful in mutantgenotypic identification in that the incorporation of the sh2-i mutantgene provides a dominant phenotype that masks the expression of the su1,se1 or sh2 mutant genes.

Specifically contemplated for use initially was the su1su1 se1se1 sh2sh2NIL triple recessive allelic combination. This specific combination waschosen to combine starch defective genes that could possibly offset therapid build up of starch that is associated with the incorporation ofthe mutant sh2-i gene into the maize genome. Backcrossing of the su1su1se1se1 sh2sh2 triple recessive NILs as recurrent parents was theninitiated using a donor sh2-i gene source provided by Dr. C. L. Hanna ofthe University of Florida (Gainesville, Fla.). (The mutant sh2-i gene,and its sequence, is shown and described in detail in U.S. Pat. No.6,184,438 B1). This donor source was test crossed to appropriate geneticlines to determine the status of the su1 and se1 genes. Test crossingresults confirmed that the donor sh2-i gene source had a genotype ofSu1Su1 Se1Se1 relative to the sugary-1 (su1) and sugary enhancer-1 (se1)genes.

Ears of corn from BC1-S1 populations were phenotypically examined.(BC1-S1 is an A&C breeding population, and the term “BC1-S1” is agenetic or plant breeder's word for point in time relating to theprogress of the final product. The final product in this example is areconstruction of the recurrent parent in which the sh2-i mutant alleleis to be added.) Only those BC1-S1 ears segregating su 1, sh2 and sh2-ikernel types were selected for continued backcrossing. The mutant sh2sh2morphological sweet corn kernel appearance is distinct from othervarieties of sweet corn in that dry seeds (kernels) appear to betranslucent, highly collapsed, and wrinkled. The mutant sh2-i sweet cornkernel phenotypes, in contrast, are smooth, heavier, well filled andnearly like that of flint field corn in appearance (having round kernelswith smooth coats). Kernel phenotypes were confirmed with molecularmarkers, as is discussed elsewhere herein, and as is shown in FIG. 10.The limitation of but two phenotypes on BC2-S1 selected ears resulted inthe verification that the kernels expressing the sh2-i gene were layeredover an su1su1 sh2sh2 genetic background.

Kernels from the above BC1-S1 selected sh2-i phenotypes (su1su1 se1se1sh2sh2 NIL inbred sh2-i conversion kernels) were then planted and testcrossed to se1se1 genetically confirmed inbreds. Molecular markergenetic confirmations were conducted concomitantly. Only plantsexhibiting homozygous se1 test cross positives and se1 molecularconfirmations were kept for continued backcrossing, selfing and testcrossing. The backcrossing, selfing and test crossing, and molecularconfirmations, were continued through six cycles. At this point, it wasconsidered that the original triple allelic su1su1 se1se1 sh2sh2 NILswere adequately converted with the inclusion of the mutant sh2-i gene intheir genomes. These phenotypically and molecularly confirmed maize earsresulted in sh2-i kernels being layered over su1su1 se1se1 geneticbackgrounds. The resultant kernel phenotype was predominantly similar tothat of kernels expressing the sh2-i gene in appearance, with thekernels being smooth, full, and relatively heavy in appearance.

Laboratory Warm and Cold Germination Testing and Organoleptic TasteTesting

Laboratory warm towel and cold soil germinations were conducted usingmethods known by those having ordinary skill in the art on the su1su1se1se1 sh2sh2 NIL inbred sh2-i conversion kernels to verify enhancedseedling performances that are associated with the mutant sh2-i gene(and the donor sh2-i). Actual cold field soil testing was conducted, aswell, for further verification of enhanced germination and seedlingvigor. The laboratory warm towel and cold soil germinations wereconducted at 45° F. (7.22° C.) and 80° F. (26.67° C.), respectively.Moisture levels were measured according to published recommendations forgermination testing protocols of the Association of Official SeedAnalysts, Inc. (AOSA) (Ithaca, N.Y.). The sweet corn kernels wereplanted in a conventional manner known by those having ordinary skill inthe art using accredited plastic germination trays sanctioned by theAOSA. In some of these tests, in particular, a sweet corn female parentline designated as AC 199Y (discussed in Examples 2 and 3), having themutant sh2-i gene incorporated into its genome, was tested against itsisogenic counterpart, which did not include the sh2-i gene in itsgenome.

Organoleptic taste testing of the su1su1 se1se1 sh2sh2 NIL kernels incomparison with kernels of its sh2-i counterpart (su1su1 se1se1 sh2sh2NIL inbred sh2-i conversion kernels) was conducted using methods knownby those having ordinary skill in the art, and very advantageouslyresulted in only slight elevations in starch synthesis, which were notconsidered to be significant. Organoleptic testing was conducted by ataste panel of experienced staff of personnel (5-8 individuals) employedby Abbott & Cobb, Inc. (Trevose, Pa.). Generally, six corn ears per itemwere selected from three replications each for any particular plantingsite. Test corn ears were maintained at ambient temperatures (rangingfrom about 80-88° F. or 26.66-31.11° C.). Pedigrees of test materialswere withheld from taste panel participant to reduce any varietal tastebias or pre-disposition.

Germination and seed viability can be determined by means of selectedwell-known university or commercial laboratory enterprises that complywith Association of Official Seed Analysis (AOSA) rules for germinationtesting protocols, such as STA Laboratories (Longmont, Colo.), MidwestLaboratories (Omaha, Nebr.), Biogenetic Services Inc. (Brookings, S.Dak.) and others.

Association of Official Seed Analysts, Inc. (AOSA): Seed TestingProtocols

As is well known by those having ordinary skill in the art, the seedtesting protocols of the Association of Official Seed Analysts, Inc.(AOSA) (Ithaca, N.Y.), an organization of member laboratories, serveuniversally as the standard for seed germination, seedling vigor, kernelorganoleptic taste and pericarp tenderness and other seed and planttesting. Members of this organization include official state, federal,university and other seed laboratories across the United States andCanada. To assure a high standard of quality, many members of the AOSAmember laboratories have acquired AOSA Certified Seed Analyst statusthrough extensive training followed by a mandatory certification testingprocess.

The primary functions of the AOSA are to: (i) establish the AOSA Rulesfor Testing Seeds which are generally adopted by most states in theUnited States as the rules for testing seeds in their respective states;(ii) contribute to the refinement and modification of the rules andprocedures for seed testing; (iii) ensure that seed testing proceduresare standardized between analysts and between laboratories; and (iv)influence and assist in enforcement of appropriate seed legislation atstate and federal levels.

The official AOSA Internet web site (aosaseed dot corn) provides directInternet links to several other official Internet web sites relating toseeds and/or plants, including those for the United States Department ofAgriculture (USDA) Seed Regulatory and Testing Programs, the Associationof American Seed Control Officials (AASCO), the Association of OfficialSeed Certifying Agencies (AOSCA), the American Seed Trade Association(ASTA), the Canadian Food Inspection Agency (CFIA), the Commercial SeedAnalysts Association of Canada (CSAAC), the National Association ofState Departments of Agriculture (NASDA), the Front Range Seed Analysis,the International Seed Testing Association (ISTA), Seed Quest SeedImagesdot corn and the Society of Commercial Seed Technologists (SCST). Theofficial AOSA Internet web site also includes an extensive product andvendor web site, which includes products directed towards seed analysiscomputer software, DNA products, markers, reagents, sequencing, Westernblots and the like.

The AOSA handbooks and rules regarding a variety of different topics,including its 2009 rules for testing seeds, a seed vigor handbook, aseed moisture handbook, and a seed technology journal, are publiclyavailable for computer downloading and/or reading from its officialInternet web site at aosaseed dot com. For example, the AOSA Seed VigorTesting Handbook is a comprehensive revision of the 1983 and 2002versions thereof that set the industry standard for seed vigor testing.Further, the Seed Technology Journal, which is also available at theInternet web site seedtechnology dot net is an international journalcontaining scientific and technological papers in all areas of seedscience and technology. The emphasis in this journal is on applied andbasic research in seed physiology, pathology and biology that may relateto seed development, maturation, germination, dormancy anddeterioration. Studies on seed production, sampling, testing,conditioning, distribution and storage are also included. Shortcommunications from seed analysts and technologists are encouraged andpublished as Seed Tech Notes. These notes include new techniques,standardization of laboratory tests and documentation of anatomical andpathological observations of seed and seedling development. The journalalso includes timely review articles in all areas of seed technologythat may relate directly to the seed industry.

Molecular Map of Samples of Inbred Parental NILs having Various TripleMutant Endosperm Allelic Combinations (FIG. 10)

FIG. 10 provides a molecular map for samples of individual inbredparental NILs having the triple mutant endosperm allelic combinationsshown below. In this molecular map:

-   -   Samples 1-2 (017 and 044, respectively) are genetically:        Su1Su1Se1Se1 sh2sh2 (including a single homozygous recessive        mutant endosperm allele).    -   Samples 3-7 (006, 007, 009, 047 and 637, respectively) are        genetically: Su1Su1se1se1 sh2sh2 (including a double homozygous        recessive mutant endosperm allelic combination).    -   Samples 8-13 (001, 046, 048, 049, 109 and 354, respectively) are        genetically: su1su1 se1se1 sh2sh2 (including a triple homozygous        recessive mutant endosperm allelic combination).    -   Sample 14 is the donor source for the mutant sh2-i gene        (received from The University of Florida, Gainesville, Fla.).        Highlighted in FIG. 10 are the purported chromosomal regions        characterized as the Se1, Su1 and Sh2 sites. Numerous molecular        markers were useful in genotypic identification. In particular,        the molecular marker designated as “umc 1551” was efficacious in        se1se1 characterizations, and the molecular marker designated as        “phi 079” was similarly useful in making su1su1 identifications.        Numerous other molecular markers that are shown in FIG. 10 were        helpful singly, or in combination, in making marker assisted        assignments.

Example 2 Production using Molecular Markers, and an se1se1/su1su1Genetic Background, and Testing of Sweet Corn Hybrid ACX SS 7501Y sh2-iFemale Parents in Combination with Male Parents having Triple RecessiveAllelic Combinations

In the experiments that are described in this example, the sweet cornhybrid NIL designated as ACX SS 7501Y having a desired triple alleliccombination was developed utilizing the sh2-i gene, the parental sweetcorn NILs having the designations shown below and molecular markers, andthen tested in the manner described below.

Construction of Hybrid ACX SS 7501Y

The mutant sh2-i gene was incorporated into a series of selected inbredparental NIL sweet corn lines in the manner that is discussed inExample 1. These inbred parental NIL sweet corn lines were chosen on thebasis of desirable horticultural and seed production criteria, as isdiscussed in Example 1 and elsewhere herein.

It was discovered through a significant amount of experimentation andorganoleptic taste and pericarp tenderness testing of various differentsweet corn hybrids produced using a female parent line and a male parentline that both included the sh2-i gene in their genomes thatunacceptable starch synthesis and buildup following the peak eatingstage of resulting hybrids developed from these lines (producing astarchy taste and sweet corn kernels that were not tender) disallowedthe process of assembling hybrids based upon the use of two sh2-iparental corn lines. The best alternative was determined by thisexperimentation and testing to be the generation of hybrids comprisedof: (i) an sh2-i conversion inbred female sweet corn line; and (ii) ahigh-quality conventional male parental sweet corn line.

In view of the results of the above-described experimentation andorganoleptic taste and pericarp tenderness testing, the inventor decidedto use the mutant sh2-i allele only in female sweet corn parent lines(i.e., that the sh2-i conversion inbred sweet corn lines would be usedonly as the female parent lines, and not as the male parent lines, incommercial maize production. This decision was based upon a number offactors, including those discussed above. One such factor for using thesh2-i conversion parents as females was based upon the fact that kernelpericarp and resulting seed coat is 2N and maternally inherited.Utilization of the sh2-i conversion parents as female parents, thus,preserves the very advantageous enhanced germination and seedling vigorcharacteristic provided by the sh2-i gene, and expressed as a functionof the female parental line. In addition, commercial seed productionyields appear to be greatly enhanced as an artifact of the superiorgermination and seedling vigor traits provided to sweet corn hybrids bythe sh2-i female seed parental lines. Sh2-i conversion inbred femaleparent sweet corn lines were determined to provide hybrids developedtherewith the multiple beneficial traits of having an enhancedgermination, an enhanced seedling vigor and an enhanced commercial seedproduction yield.

In view of the above, the genotypes of the male parental lines selectedfor use in hybrid combinations with the sh2-i conversion female lineswere chosen based upon their ability to provide desirable horticulturalqualities to sweet corn hybrids produced using such lines, but primarilyby its contribution to producing sweet corn hybrids having a high eatingquality, such as a sweet taste and a tender kernel pericarp. Thus, maleparental genotypes were utilized that contained su1su1 se1 set sh2sh2triple honiozygous recessive mutant endosperm allelic combination. Thetriple homozygous recessive lines su1su1 se1se1 sh2sh2 generally tend tobe proportionately higher in sucrose as compared to similar doublehomozygous recessive mutant endosperm lines, such as su1su1 Se1 Se1sh2sh2 or su1su1 sh2sh2, and thus were determined to be more desirablefor use as the male parent lines for producing hybrids in the mannersthat are described herein. (W. F. Tracy, A. R. Hellaurer (ED) SpecialtyCorns, 147-187 (CRS Press, Boca Raton, Fla. (1994).) (The doublehomozygous recessive su1su1 sh2sh2 genotypes, however, including su1su1Se1Se1 sh2sh2, as well as the triple homozygous recessive su1su1 se1se1sh2sh2 genotypes, which are characteristic of male sweet corn parentlines that are described herein, are also desirable for use in theproduction of sweet corn hybrids in that they are exceptionally high insugar with very tender kernel pericarps. The su1su1 sh2sh2 homozygousrecessive genotypic components (i.e., the double homozygous recessivesu1su1 and sh2sh2 mutant endosperm alleles present in both the abovedouble and triple homozygous recessive endosperm mutant alleliccombination) of all of these male sweet corn parent lines providesignificant holding abilities (retention of higher sugars and tenderkernel pericarps) in hybrid combination with the female sh2-i conversionsweet corn lines that are described herein (i.e., resulting hybridsinclude these traits).)

One resulting sweet corn hybrid containing the sh2-i gene of particularinterest was designated as hybrid ACX SS 7501Y. This hybrid linediffered in a beneficial manner from other hybrids prepared in thisexample in performance relative to geographical growing regions and/ormarket requirements, and its characteristics are described in detail inthe Characteristics Table (Hybrid Sweet Corn lines) that is set forthhereinabove prior to Example 1. The Characteristics Table (ParentalSweet Corn lines) describes the characteristics in detail of the parentsweet corn lines that were employed to develop this hybrid line.

Laboratory Warm and Cold Germination Testing and Organoleptic Taste andPericarp Tenderness Testing

Organoleptic evaluation of sweet corn hybrids that were assembled in theabove fashion, including hybrid ACX SS 7501Y, were determined usingmethods that are known by those of ordinary skill in the art, as isdiscussed in great detail in Example 1. Such hybrids were shown toprovide significant benefits to corn growers in that germination,seedling vigor, and overall crop productivity was elevated in comparisonto the same or similar sweet corn varieties not including the sh2-i genein their genomes. Additionally, consumers of these sweet corn hybridproducts receive valuable benefits relating to product eating quality(sweet taste and kernel pericarp tenderness) and extended shelf life.

FIG. 7 illustrates the comparative organoleptic sweetness (sugar) scoresamong three sweet corn varieties over a period of seven days directlyfollowing the peak eating stage. The commercial variety Passion is soldand distributed through Monsanto (St. Louis, Mo.). The commercialvariety Beyond is sold and distributed by Abbott and Cobb, Inc.(Trevose, Pa.), and has served as a standard, primarily in the Southeastcommercial shipping markets. The variety designated as ACX SS 7501Y is ahybrid variety that was produced in accordance with Examples 1 and 2using a female parent line designated as AC 199Y and a male parent linedesignated as AC 195Y. The organoleptic sweetness (sugar) scores rangefrom 1 (very little sweetness with a considerable starch taste) to 10(sweet with little or no starch taste). FIG. 7 shows that the ACX SS7501Y hybrid corn variety very advantageously maintained an organolepticsweetness score above both the Passion and Beyond corn varieties at alltimes during this 7-day period and, in contrast with the Passion andBeyond corn varieties, maintained a score of 10 on days 1 and 2 pastprime eating stage. FIG. 7 also shows that the ACX SS 7501Y hybrid cornvariety had an organoleptic sweetness score of almost 8 on day 7 (incomparison with a score of about 5 for Passion, and a score of about 3for Beyond). Over the seven day testing period, the ACX SS 7501Y hybridcorn variety held, and maintained, very high sweetness levels comparedto the comparison varieties of Passion and Beyond.

FIG. 8 shows the comparative organoleptic pericarp and tenderness scoresamong the same three sweet corn varieties that are shown in FIG. 7 overa period of seven days directly following the peak eating stage. Theorganoleptic pericarp and tenderness scores range from 1 (very toughpericarp) to 10 (very tender pericarp). FIG. 8 shows that the ACX SS7501Y hybrid corn variety very advantageously maintained an organolepticpericarp and tenderness score above both the Passion and Beyond cornvarieties at all times during this 7-day period. It also shows that theACX SS 7501Y hybrid corn variety had an organoleptic pericarp andtenderness score of about 7 on day 7 (in comparison with a score ofabout 5 for Passion, and a score of about 2 for Beyond). Over the sevenday testing period, the ACX SS 7501Y hybrid corn variety held, andmaintained, very high pericarp tenderness levels compared to thecomparison varieties of Passion and Beyond. The ACX SS 7501Y hybrid cornvariety was observed to be very tender, and to have an excellent holdingability. This hybrid line differed in a beneficial manner from otherhybrids prepared in this example in performance relative to geographicalgrowing regions and/or market requirements, and its characteristics aredescribed in detail in the Characteristics Table (Hybrid Sweet Cornlines) that is set forth hereinabove prior to Example 1.

Example 3 Production using Molecular Markers, and a se1se1/su1su1Genetic Background, and Testing of Sweet Corn Hybrids ACX SS 7078Y andACX SS 7403RY sh2-i Female Parents in Combination with Male Parentshaving Triple Recessive Allelic Combinations

In the experiments that are described in this example, additional sweetcorn hybrid NILs having desired triple allelic combinations weredeveloped utilizing the sh2-i gene and molecular markers, and thentested in the manner described below.

Construction of Hybrids ACX SS 7078Y and ACX SS 7403RY

The additional sweet corn hybrids were assembled according to the samebreeding procedures that are described in Examples 1 and 2 for sweetcorn hybrid ACX SS 7501Y. These sweet corn varieties were assembled bylayering the sh2-i gene over se1se1 and su1su1 genetic backgroundsutilizing molecular markers, as has been described previously herein.

Two resulting sh2-i sweet corn hybrids of particular interest weredesignated as ACX SS 7078Y and ACX SS 7403RY, and were developed usingthe parental sweet corn NILs having the designations shown below. Thesehybrid lines differed in a beneficial manner from other hybrids preparedin this example in performance relative to geographical growing regionsand/or market requirements, and their characteristics are described indetail in the Characteristics Table (Hybrid Sweet Corn lines) that isset forth hereinabove prior to Example 1. The Characteristics Table(Parental Sweet Corn lines) describes the characteristics in detail ofthe parent sweet corn lines that were employed to develop these hybridlines.

Sweet corn hybrid ACX SS 7078Y, which has the sh2-i mutant alleleincorporated into its genome, is an isogenic conversion of the Abbottand Cobb, Inc. commercially-available hybrid designated ACX 1073Y, whichdoes not have the sh2-i mutant allele incorporated into its genome.Similarly, sweet corn hybrid ACX SS 7403RY, which has the sh2-i mutantallele incorporated into its genome, is an isogenic conversion of theAbbott and Cobb, Inc. commercially-available variety designated ACX7473RY, which also does not have the sh2-i mutant allele incorporatedinto its genome. In both sh2-i gene containing hybrid developments,these two isogenic sweet corn hybrids were found to be nearly identicalfor all horticultural and morphological characteristics, when identifiedusing methods that are described herein and/or are known by those havingordinary skill in the art. The identification methods and differentialcharacterization of isogenic comparisons and nomenclature are well knownby those having ordinary skill in the plant breeding and similar arts,as well as in scientific communities generally.

Laboratory Warm and Cold Germination Testing and Organoleptic Taste andPericarp Tenderness Testing

Table 3 below provides data resulting from actual comparisons oflaboratory warm and cold germination data, as well as organoleptic tasteand pericarp tenderness tests, for the sweet corn varieties ACX 1073Y,ACX SS 7078Y, ACX 7473RY and ACX SS 7403RY. Germination data reflect themean of three replications of 100 kernels each, and germination scoresnot followed by the same letter are significantly different at the 0.05probability level via the Duncan's New Multiple Range Test. In Table 3(and in other tables that are set forth herein), the “a” and “b”designations indicate that the mean values are significantly differentat the 0.05 probability level. (The same is true of a “c” designationappearing in other tables set forth herein.) The organoleptic testsregarding sweetness and pericarp tenderness are described previouslyherein.

Table 3 shows that, in both cases in which the sh2-i mutant gene wasadded to the sweet corn genomes (hybrids ACX SS 7078Y and ACX SS7403RY), the isogenic hybrid comparison warm and cold laboratory scoresvery advantageously were elevated in comparison with the scores of thecommercially-available sweet corn varieties which do not include thesh2-i mutant gene in their genomes (ACX 1073 and ACX 7473RY), which isindicative of enhanced field emergence and vigor. Table 3 also showsthat the two sweet corn varieties including the mutant sh2-i gene intheir genomes (hybrids ACX SS 7078Y and ACX SS 7403RY) veryadvantageously retained their sweetness (sugar levels) significantlylonger (having scores of 8 on Day 7 in the seven day period immediatelyfollowing the prime eating stage) than the two sweet corn varieties thatdid not include this sh2-i gene in their genomes (having scores of 6 or5 on Day 7), and had pericarps that retained their tendernesssignificantly longer (having scores of 7 on Day 7 in the seven dayperiod immediately following the prime eating stage) than the two sweetcorn varieties that did not include this sh2-i gene in their genomes(having scores of 6 or 5 on Day 7). The organoleptic sweetness andpericarp tenderness scores that are present in Table 3 veryadvantageously demonstrate acceptable eating quality levels along withconcomitantly desirable shelf life and holding abilities for hybrids ACXSS 7078Y and ACX SS 7403RY.

TABLE 3 Isogenic Comparisons of Sweet Corn Varieties Including, or notIncluding, the sh2-i Gene Organoleptic Sweetness Organoleptic PericarpWarm Cold Score Tenderness Score Variety Germination Germination Day 1Day 4 Day 7 Day 1 Day 4 Day 7 ACX 92a 59b 9 8 6 9 8 6 1073Y (Does notContain sh2-i Gene) ACX 98a 91a 9 8 8 9 8 7 SS 7078Y (Contains sh2-iGene) ACX 91a 62b 9 7 5 8 6 5 7473RY (Does not Contain sh2-i Gene) ACX97a 94a 10  8 8 9 7 7 SS 7403RY (Contains sh2-i Gene)

Example 4 Production without using Molecular Markers, and ase1se1/su1su1 Genetic Background, and Testing of Sweet Corn Hybrids AC151Y and AC 188W sh2-i Female Parents in Combination with Male Parentshaving Double or Triple Recessive Allelic Combinations

In the experiments that are described in this example, additional sweetcorn hybrid NILs having double and triple allelic combinations weredeveloped utilizing the sh2-i gene, but without the use of molecularmarkers, and then tested in the manner described below.

Construction of Hybrids AC 151Y and AC 188W

These additional sweet corn hybrids were assembled according to the samebreeding procedures that are described in Examples 1, 2 and 3, with theexceptions that the hybrid lines containing the mutant sh2-i gene intheir genomes were not assembled utilizing molecular markers, aspreviously described herein (Examples 1, 2 and 3), and did notincorporate se1se1/su1su1 alleles.

Two resulting sweet corn sh2-i hybrids of particular interest weredesignated AC 151Y and AC 188W, and were developed using the parentalsweet corn NILs having the designations shown below. These hybrids weredeveloped from sh2-i female conversion parent lines not containing thesu1 and se1 genes to test eating quality in comparison with sh2-i femaleparental inbreds constructed utilizing the incorporation of the su1 andse1 genes. It was also considered important to test the effect of usingdouble and triple homozygous recessive male lines with the sh2-iconversion females lines that were not constructed utilizing sequentiallayering over the su1 and se1 genes.

These hybrid lines differed in a beneficial manner from other hybridsprepared in this example in performance relative to geographical growingregions and/or market requirements.

With both hybrid lines AC 151Y and AC 188W, female parent lines wereconstructed using the mutant sh2-i gene not in combination with the su1and/or se1 genes. The hybrid AC 151Y line, however, was developed usinga triple recessive male parent line, whereas the hybrid AC 188W line wasdeveloped using a double recessive male parent line.

The hybrid line AC 151Y (an sh2-i conversion inbred including the mutantsh2-i gene in its genome) is an sh2-i gene isogenic conversion of, andcomparison to, an Abbott and Cobb, Inc. hybrid designated as ACR 5132Y(not including the mutant sh2-i gene in its genome). The onlysignificant genetic difference between these two hybrid lines in thisexample is the presence of the mutant sh2-i gene in hybrid line AC 151Yand the absence of the sh2-i gene in hybrid line ACR 5132Y.

The hybrid line AC 188W (including the mutant sh2-i gene in its genome)is an sh2-i gene isogenic conversion of, and comparison to, an Abbottand Cobb, Inc. hybrid designated as ACR 5147W (not including the mutantsh2-i gene in its genome). Again, the only significant geneticdifference between these two hybrid lines in this example is thepresence of the mutant sh2-i gene in hybrid line AC 188W and the absenceof the sh2-i gene in hybrid line ACR 5147W.

Laboratory Warm and Cold Germination Testing and Organoleptic Taste andPericarp Tenderness Testing

Table 4 below provides data resulting from actual comparisons oflaboratory warm and cold germination data, as well as organoleptic tasteand pericarp tenderness tests, for the sweet corns AC 151Y, ACR 5132Y,AC 188W and ACR 5147W. Germination scores are means of threereplications of 100 kernels each. Laboratory scores not followed by thesame number are significantly different at the 0.05 probability levelvia the Duncan's New Multiple Range Test. The organoleptic testsregarding sweetness (sugar content) and pericarp tenderness aredescribed previously herein.

Table 4 shows that, in both cases in which the sh2-i mutant gene wasadded to the sweet corn genomes (hybrids AC 151Y and AC 188W), theisogenic hybrid comparison warm and cold laboratory scores wereelevated, which is indicative of enhanced field emergence and vigor.However, Table 4 also shows that these two sweet corn varietiesincluding the mutant sh2-i gene did not retain their sweetness longer(having scores of 2 and 3 on Day 7 in the seven day period immediatelyfollowing the prime eating stage) in comparison with the two sweet cornvarieties that did not include this gene (hybrids ACR 5132Y and ACR5147W) (having scores of 5 and 7 on Day 7), and had pericarps that didnot retain their tenderness longer (having scores of 1 and 2 on Day 7 inthe seven day period immediately following the prime eating stage) incomparison with the two sweet corn varieties that did not include thisgene (having scores of 3 and 2 on Day 7). The sweetness and pericarptenderness scores that are present in Table 4 suggest that an insertioninto inbred lines of the mutant sh2-i gene without a sequential layeringagainst a genetic background of se1se1 and su1su1, as has been describedpreviously herein, results in less efficacious and some detrimentaleffects in connection with overall eating quality (sweetness andpericarp tenderness).

In view of the above, it is considered to be very desirable (or, in somecases, as may readily be determined by those having ordinary skill inthe art, even necessary) to assemble and direct the construction ofmutant sh2-i gene materials in the manner that has been describedpreviously herein in order to obtain maximum benefits desired by plantgrowers and consumers (i.e., using molecular markers and an se1se1 andsu1su1 genetic background).

TABLE 4 Isogenic Comparisons of Sweet Corn Varieties Including, or notIncluding, the sh2-i Gene Organoleptic Sweetness Organoleptic PericarpWarm Cold Score Tenderness Score Hybrid Germination Germination Day 1Day 4 Day 7 Day 1 Day 4 Day 7 AC 151Y 99a 90a 8 5 2 7 3 1 (Containssh2-i Gene) ACR 5132Y 98a 82b 8 6 5 8 5 3 (Does not Contain sh2-i Gene)AC 188W 96a 88b 9 4 3 6 4 2 (Contains sh2-i Gene) ACR 5147W 88b 47c 10 8 7 9 4 2 (Does not Contain sh2-i Gene)

Example 5 Comparison of Various Physical Characteristics of Sweet CornHybrids ACX SS 7501Y, ACX SS 7078Y and ACX SS 7403RY sh2-i FemaleParents in Combination with Male Parents having Triple Recessive AllelicCombinations

In the experiments that are described in this example, the physicalcharacteristics of sweet corn hybrids ACX SS 7501Y, ACX SS 7078Y and ACXSS 7403RY, all of which include the mutant sh2-i gene in their genomes,and very advantageously have the numerous and varied beneficial benefitsthat are described herein, were examined and compared using methods thatare well known by those having ordinary skill in the art. Most of theexaminations and comparisons were conducted using conventional maizevisual observation, counting and measurement techniques, all of whichare known by those having ordinary skill in the art. The results ofthese examinations and comparisons are set forth in Table 5 below. Ineach column in Table 5, the data for sweet corn hybrid ACX SS 7501Y isfollowed by the data for sweet corn hybrid ACX SS 7078Y, which isfollowed by the data for sweet corn hybrid ACX SS 7403RY. In Table 5,the term “Maturity” refers to the number of days that elapsed from thetime that the hybrid was planted until it matured. The diseaseresistance ratings were performed by Dr. Jerald Pataky at the Universityof Illinois (Urbana-Champaign, Ill.) using well-known methods in anindependently and controlled plant pathological environment.

TABLE 5 Comparative Data for Three Sweet Corn Hybrids Including theMutant Sh2-i Gene ROW EAR EAR HYBRID MATURITY COUNT LENGTH SHAPE ACX SS76 days 16 8.0″ Cylindrical 7501Y ACX SS 78 days 16-18 8.25″ Cylindrical7078Y ACX SS 75 days 16-18 8.25″ Cylindrical 7403RY DISEASE EAR PACKAGEKERNEL COLOR PLANT SIZE RESISTANCE Excellent flags, husk Yellow MediumIntermediate color resistance to northern corn leaf blight and commonrust Very good husk Yellow Medium/Tall None claimed color and lengthDark green husk and Yellow Medium Intermediate flags resistance tonorthern corn leaf blight, resistance to multiple races of common rust

Deposits Deposits made with the American Type Culture Collection (ATCC)in Connection with Examples 1-5

The seed deposits discussed below, which relate to Examples 1-5, havebeen made with the American Type Culture Collection (ATCC) (Manassas,Va., USA) by Applicant Bryant J. Long on behalf of assignee Abbott &Cobb, Inc. (Wellington, Fla.) for the Zea mays, sweet corn hybridsidentified below having desired triple allelic combinations, which eachinclude the sh2-i gene in their genomes, and the corresponding Zea mays,sweet corn parent lines that were employed to develop these sweet cornhybrids. In the tables shown below, in connection with the sugary (su1),sugary enhancer-1 (se1) and shrunken-2 (sh2) genes, the designation “D”indicates that the male parent line included a double recessive alleliccombination of these three genes, and the designation “T” indicates thatthe male parent line included a triple recessive allelic combination ofthese three genes.

Example Deposit Allelic Hybrid Parent Lines No. Made Combination ACX SS7501Y AC 199Y (Female) × AC 195Y (Male) 2 Yes* T ACX SS 7078Y AC 199Y(Female) × AC 128Y (Male) 3 Yes* T ACX SS 7403RY AC 199Y (Female) × AC215Y (Male) 3 Yes* T AC 151Y AC 103Y (Female) × AC 107Y (Male) 4 No T AC188W AC 106W (Female) × AC 163W (Male) 4 No D *All three lines (the twoparents and the hybrid identified) have been deposited.

ATCC Patent Identification Reference Deposit Used by Depositor/InventorDesignation Quantity Received (1) Sweet Corn, Zea mays: PTA-10507 100packets/25 seeds ACX SS 7501Y in each packet (2) Sweet Corn, Zea mays:PTA-10506 100 packets/25 seeds ACX SS 7078Y in each packet (3) SweetCorn, Zea mays: PTA-10508 100 packets/25 seeds ACX SS 7403RY in eachpacket (4) Sweet Corn, Zea mays: PTA-10981 100 packets/25 seeds AC 128Yin each packet (5) Sweet Corn, Zea mays: PTA-10982 100 packets/25 seedsAC 195Y in each packet (6) Sweet Corn, Zea mays: PTA-10983 100packets/25 seeds AC 199Y in each packet (7) Sweet Corn, Zea mays:PTA-10984 100 packets/25 seeds AC 215Y in each packet

Hybrid Lines

Seeds of sweet corn, Zea mays, hybrids ACX SS 7501Y, ACX SS 7078Y andACX SS 7403RY (100 packets for each of these three hybrids, with 25seeds in each packet) were deposited on behalf of Abbott & Cobb, Inc.with the American Type Culture Collection (ATCC) (10801 UniversityBoulevard, Manassas, Va., 20110-2209, United States of America) underThe Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms on Nov. 30, 2009, and were given ATCC Patent DepositDesignations PTA-10507, PTA-10506 and PTA-10508, respectively, by theATCC. The deposited seeds were tested on Dec. 14, 2009 by the ATCC and,on that date, they were viable. The depositor was Abbott & Cobb, Inc.,11460 Fortune Circle, Wellington, Fla., 33414, United States of America.

Parent Lines

Seeds of the sweet corn, Zea mays, female and male parent lines thatwere employed to develop sweet corn, Zea mays, hybrid ACX SS 7501Y (100packets, with 25 seeds in each packet), designated AC 199Y (female) andAC 195Y (male), were deposited on behalf of Abbott & Cobb, Inc. with theAmerican Type Culture Collection (ATCC) (10801 University Boulevard,Manassas, Va., 20110-2209, USA) under The Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms on May 19,2010, and were given ATCC Patent Deposit Designations PTA-10983 andPTA-10982 by the ATCC, respectively. The deposited seeds were tested onJun. 1, 2010 by the ATCC and, on that date, they were viable.

Seeds of the sweet corn, Zea mays, female and male parent lines thatwere employed to develop sweet corn, Zea mays, hybrid ACX SS 7078Y (100packets, with 25 seeds in each packet), designated AC 199Y (female) andAC 128Y (male), were deposited on behalf of Abbott & Cobb, Inc. with theAmerican Type Culture Collection (ATCC) (10801 University Boulevard,Manassas, Va., 20110-2209, USA) under The Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms on May 19,2010, and were given ATCC Patent Deposit Designations PTA-10983 andPTA-10981, respectively, by the ATCC. The deposited seeds were tested onJun. 1, 2010 by the ATCC and, on that date, they were viable.

Seeds of the sweet corn, Zea mays, female and male parent lines thatwere employed to develop sweet corn, Zea mays, hybrid ACX SS 7403RY (100packets, with 25 seeds in each packet), designated AC 199Y (female) andAC 215Y (male), were deposited on behalf of Abbott & Cobb, Inc. with theAmerican Type Culture Collection (ATCC) (10801 University Boulevard,Manassas, Va., 20110-2209, USA) under The Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms on May 19,2010, and were given ATCC Patent Deposit Designations PTA-10983 andPTA-10984, respectively, by the ATCC. The deposited seeds were tested onJun. 1, 2010 by the ATCC and, on that date, they were viable.

Certificates of Deposit have been received from the American TypeCulture Collection (ATCC) for each of the deposits that are describedabove.

Example 6 Construction and Testing of Sweet Corn Parental Inbred Line AC157Y and Parental Near Isogenic Line (NIL) AC 157Y-i sh2-i FemaleParents in Combination with Male Parents not having Double or TripleRecessive Allelic Combinations

In the experiments that are described in this example, in order toattempt to solve the problem of manipulating the regulation ofcarbohydrate accumulation, and pericarp tenderness, in sweet cornvarieties containing the mutant sh2-i gene, appropriate parental nearisogenic lines (NILs) were constructed and tested, using methods thathave previously been described herein or that are described below and/orare known by those having ordinary skill in the art, of the followinggenotypic combinations:

Female Parent Lines (NILs)

-   -   AC 157Y: Su1Su1Se1Se1 sh2sh2    -   AC 157Y-i: Su1Su1 Se1Se1 sh2-i sh2-i

Construction of Parental NILs

The mutant sh2-i gene was incorporated into selected inbred parent lineshaving the above gene combinations. Such parent inbreds were alsoselected on the basis of desirable horticultural and seed productioncriteria, including those that are discussed herein. It was discoveredthrough a significant amount of organoleptic experimentation and testingthat unacceptable starch synthesis and starch buildup occurredimmediately following the peak eating stage with some sweet corn hybridsproduced using the sh2-i conversion inbreds described above,particularly those hybrids produced using two sh2-i conversion inbredparent lines (i.e., two parental lines that both include a mutant sh2-igene in their genomes), thus disallowing an assembly of hybrids usingtwo sh2-i parent lines. Through this organoleptic experimentation andtesting, it was eventually determined that a potentially desirablegenotype to use for female sweet corn parent lines was Su1Su1Se1Se1sh2-i sh2-i, and that the utilization of male parent lines having oneset of homozygous recessive mutant alleles (Su1Su1 Se1Se1 sh2sh2) couldprove beneficial. The corresponding hybrid genotype is of the followingcomposition:

Female Parent Line

In view of the above and below, the inventor decided to use the sh2-iconversion inbred sweet corn lines only as the female parents (i.e., notas the male parents) in commercial maize production. This decision wasbased upon a number of factors, including those discussed above andpreviously herein. One such factor for using the sh2-i conversion inbredparents as females was based upon the fact that kernel pericarp andresulting seed coat is 2N and maternally inherited. Utilization of thesh2-i conversion inbreds as female parents, thus, preserves the veryadvantageous enhanced germination and seedling vigor characteristicprovided by the sh2-i gene, and expressed as a function of the femaleparental line. In addition, commercial seed production yields appear tobe greatly enhanced as an artifact of the superior germination andseedling vigor traits provided to sweet corn hybrids by the sh2-i femaleseed parental lines. Sh2-i conversion inbred female parent sweet cornlines were determined to provide hybrids developed therewith themultiple beneficial traits of having an enhanced germination, anenhanced seedling vigor and an enhanced commercial seed productionyield.

Backcrossing of various commercial inbred lines was initiated using thedonor sh2-i gene source provided by Dr. C. L. Hanna of the University ofFlorida (Gainesville, Fla.). This donor source was test crossed toappropriate genetic lines to determine the status of the su1 and se1genes. Test crossing results confirmed that the donor sh2-i gene sourcehad a genotype of Su1Su1Se1Se1 relative to the sugary-1 (su1) and sugaryenhancer-1 (se1) genes.

Ears of corn from BC1-S1 populations were phenotypically examined.(BC1-S1 is an Abbott and Cobb, Inc. breeding population, and the term“BC1-S1” is a genetic or plant breeder's word for point in time relatingto the progress of the final product. The final product in this exampleis a reconstruction of the recurrent parent in which the sh2-i mutantallele is to be added.) Only kernels expressing a hard seed coatcharacteristic of flint or dent corns were saved and utilized in furtherbackcrossing cycles. A minimum of five backcross cycles involvingrecurrent parents were performed to insure adequate reconstitution ofcommercial sweet corn lines incorporating the mutant sh2-i gene therein.The resultant kernel phenotype was predominantly similar to that of thesh2-i appearance, being smooth, full and relatively heavy. (The mutantsh2sh2 morphological sweet corn kernel appearance is distinct from othervarieties of sweet corn in that dry seeds (kernels) appear to betranslucent, highly collapsed, and wrinkled. The mutant sh2-i sweet cornkernel phenotypes, in contrast, are smooth, heavier, well filled andnearly like that of flint field corn in appearance (having round kernelswith smooth coats)). Kernel phenotypes were confirmed with molecularmarkers, as is discussed elsewhere herein, and as is shown in FIG. 10.

Laboratory warm and cold soil germination testings were conducted on thesh2-i conversion lines to verify enhanced seedling performancesassociated with the donor sh2-i source. Actual cold field soil testingwas conducted for further verification of enhanced germination andseedling vigor. Organoleptic taste testing of the resulting Su1Su1 sh2-ish2-i conversion lines and the recurrent comparison Su1Su1 sh2sh2 lineswere conducted for verification of sugar, pericarp and holding ability.

One sh2-i conversion female parent line of particular interest wasdesignated as AC 157Y-i. The Characteristics Table (Parental Sweet Cornlines) set forth hereinabove prior to Example 1 describes thecharacteristics in detail of this particular parent sweet corn line.

Germination and Seedling Vigor Testing of Female Parent Lines

Table 6 and Table 7 below show comparative laboratory warm and coldgermination values, and field soil germination and seedling vigor data,respectively for: (i) a commercial “supersweet” (Su1Su1 sh2sh2) inbredparent line designated AC 157Y (not including the sh2-i gene in itsgenome); and (ii) the NIL female (Su1Su1 sh2-i sh2-i) inbred parent line(an sh2-i conversion inbred) designated AC 157Y-i (including the sh2-igene in its genome). These parent lines are the same, with the exceptionthat one does not include the sh2-i gene in its genome, and the otherincludes the sh2-i gene in its genome. These tests were performed in thesame manner as is previously described elsewhere. Means not followed bythe same letter are significantly different at the 0.05 probabilitylevel via the Duncan's Multiple Range Statistical Test.

Both the very significant advantage of the laboratory cold germinationdata (Table 6), as well as the very desirable phenotypic seedmorphological distinctiveness, of female sh2-i conversion inbred parentline AC 157Y-i in comparison with inbred parent line AC 157Y is clearlyapparent from this data. Further, the AC 157Y-i conversion inbred parentline very desirably exhibits a smooth hard seed coat as compared withthe very undesirable shrunken, collapsed appearance of the seed of theconventional inbred parent line AC 157Y. Moreover, the data for fieldsoil germination and seedling vigor performance (Table 7) show asignificantly enhanced, and much more desirable, field soil germinationand seedling vigor for the AC 157Y-i parent sh2-i conversion inbred linein comparison with the AC 157Y parent inbred line.

TABLE 6 Warm Germination Cold Germination Inbred Parent Line(Percentage)* (Percentage)* AC 157Y 94a 71a (not including an sh2-igene) AC 157Y-i 98a 94b (including an sh2-i gene) (Female Parent Line)*The scores represent the mean of three replications of 100 kernelseach.

TABLE 7 Field Soil Germination Inbred Parent Line Percentage* SeedlingVigor** AC 157Y 86a 3a (not including an sh2-i gene) AC 157Y-i 97b 5b(including an sh2-i gene) (Female Parent Line) *The scores represent themean of three replications of 100 kernels each. Soil temperaturesaveraged approximately 50° F. **Seedling vigor was rated on a scale of1-5, with 5 representing excellent seedling vigor and 1 representingpoor seedling vigor.

Male Parent Line

In view of the above, the genotype of the male parental lines selectedfor use in hybrid combinations with the sh2-i conversion female lineswas chosen based upon its ability to provide desirable horticulturalqualities to sweet corn hybrids produced using such lines. Thus, maleparental line genotypes were utilized that included more conventional“supersweet” genetics of the composition Su1Su1Se1Se1 sh2sh2. The malesweet corn lines of this class that are set forth in this example aretypically inbred materials that are commercially available from Abbottand Cobb, Inc. (see Example 7). These male parental genotypes have butone set of homozygous recessive mutant alleles. The sweetness andtenderness of kernels of such male parental lines are generallysignificantly less in comparison with the sweetness and tenderness ofmale parental sweet corn lines including a genetic compositioncontaining two or three homozygous recessive mutant genes, such asSu1Su1 se1 se1 sh2sh2 or su1su1se1se1 sh2sh2, respectively. With respectto possible different desirable sweet corn traits, such as an enhancedvigor, this male parent line genotype was chosen as a result of itsability to provide hybrids developed therefrom with: (i) desirablehorticultural plant and ear characteristics; and (ii) high yieldingcapabilities.

Organoleptic Testing of Female Parent Lines (Sweetness and PericarpTenderness)

FIG. 11 is a line graph that illustrates comparative organolepticaverage sugar (sweetness) scores for the sweet corn inbred parent lineAC 157Y (not including the sh2-i gene in its genome) and the NIL femaleinbred parent line (sh2-i conversion inbred) designated AC 157Y-i(including the sh2-i gene in its genome) in the seven day periodimmediately following the prime eating stage (at a level ofapproximately 75% moisture). FIG. 11 illustrates the comparativeorganoleptic sugar levels among these two sweet corn inbred parent linesover this seven day period of time. The organoleptic sweetness scoresranged from 1 (very little sweetness with a considerable starch taste)to 10 (sweet with little or no starch taste). FIG. 11 shows that, ondays 1 and 2 past the prime eating stage, female parent line AC 157Ysh2-i had comparatively high organoleptic sweetness scores (about 8.5 onday 1 and about 7.5 on day 2), which were the same scores shown byparent line AC 157Y. The organoleptic sweetness score for female parentline AC 157Y-i on day 3 was somewhat higher than 5, and on days 4 and 5was about 2, and on days 6 and 7 was about 1.5. The organolepticsweetness score for parent line AC 157Y on day 3 was about 7, on day 4was about 5, on days 5 and 6 was about 4, and on day 7 was somewhathigher than 3.

FIG. 12 is a line graph that shows the comparative organoleptic pericarptenderness scores for the same sweet corn inbred parent lines in theseven day period immediately following the prime eating stage (at alevel of approximately 75% moisture). The organoleptic tenderness scoresranged from 1 (very tough pericarp) to 10 (very tender pericarp). FIG.12 shows that the AC 157Y-i female parent sweet corn line maintained anorganoleptic pericarp tenderness score from about 6 to about 7 in days1, 2 and 3 past the prime eating stage, and scores of about 5 on day 4,about 4 on day 5, about 2 on day 6, and about 1.5 on day 7. Similarscores are shown for parent line AC 157Y on days 1, 2 and 3 past theprime eating stage. The AC 157Y-i parent sweet corn line was ofcomparative initial tenderness to the AC 157Y sweet corn parent line,but with a noticeable decline over time.

Example 7 Production using Molecular Markers and Testing of Sweet CornNear Isogenic Line (NIL) Hybrid ACX SS 1082Y and Hybrid ACX SS 1082Ysh2-i Specific Allelic Combinations

In the experiments that are described in this example, sweet corn hybridNILs having specific allelic combinations were developed utilizing thesh2-i gene and molecular markers, and then tested in the mannerdescribed below.

Construction of Hybrid ACX SS 1082Y sh2-i

Sweet corn hybrids having specific allelic combinations were assembledaccording to the same breeding procedures that are previously describedherein. One resulting sh2-i sweet corn hybrid NIL (an sh2-i conversioninbred) of particular interest was designated as ACX SS 1082Y sh2-i.,and was developed using the AC 157Y-i female parental sweet corn NILline that is discussed in Example 6 along with the male parental line AC144Y (an inbred line that is commercially available from Abbott andCobb, Inc.), both of which are also identified below.

Germination and Seedling Vigor Testing

Table 8 and Table 9 below show comparative laboratory warm and coldgermination values, and field soil germination and seedling vigor data,respectively for: (i) a sweet corn hybrid NIL designated ACX 1082Y (notincluding the sh2-i gene in its genome); and (ii) the sweet corn hybridNIL designated ACX SS 1082Y sh2-i (an sh2-i conversion inbred includingthe sh2-i gene in its genome). These hybrids are the same, with theexception that one does not include the sh2-i gene in its genome, andthe other includes the sh2-i gene in its genome. These tests wereperformed in the same manner as has previously been described herein.Means not followed by the same letter are significantly different at the0.05 probability level via the Duncan's Multiple Range Statistical Test.

Both the very significant advantage of the laboratory cold germinationdata (Table 8), as well as the very desirable phenotypic seedmorphological distinctiveness, of sweet corn hybrid ACX SS 1082Y sh2-i(including the sh2-i gene in its genome) in comparison with sweet cornhybrid ACX 1082Y (not including the sh2-i gene in its genome) is clearlyapparent from this data. Further, sweet corn hybrid ACX SS 1082Y sh2-ivery desirably exhibits a smooth hard seed coat as compared with thevery undesirable shrunken, collapsed appearance of the seed of sweetcorn hybrid ACX 1082Y. Moreover, the data for field soil germination andseedling vigor performance (Table 9) show a significantly enhanced, andmuch more desirable, field soil germination and seedling vigor for sweetcorn hybrid ACX SS 1082Y sh2-i in comparison with sweet corn hybrid ACX1082Y.

TABLE 8 Warm Germination Cold Germination Hybrid (Percentage)*(Percentage)* ACX 1082Y 93a 77a (NIL not including an sh2-i gene) ACX SS1082Y sh2-i 99a 96b (NIL including an sh2-i gene) *The scores representthe mean of three replications of 100 kernels each.

TABLE 9 Field Soil Germination Hybrid (Percentage)* Seedling Vigor ACX1082Y 90a 3a (NIL not including an sh2-i gene) ACX SS 1082Y sh2-i 97b 5b(NIL including an sh2-i gene) *The scores represent the mean of threereplications of 100 kernels each. Soil temperatures averagedapproximately 50° F. **Seedling vigor was rated on a scale of 1-5, with5 representing excellent seedling vigor and 1 representing poor seedlingvigor.

Organoleptic Testing (Sweetness and Pericarp Tenderness)

FIG. 13 is a line graph that shows organoleptic average sugar(sweetness) scores for the sweet corn varieties Beyond (not includingthe sh2-i gene), Passion (not including the sh2-i gene) and hybrid ACXSS 1082Y sh2-i (an sh2-i conversion inbred including the sh2-i gene) inthe seven day period immediately following the prime eating stage (at alevel of approximately 75% moisture). It illustrates the comparativeorganoleptic sugar levels among these three sweet corn varieties overthis seven-day period of time. The organoleptic sweetness scores rangedfrom 1 (very little sweetness with a considerable starch taste) to 10(sweet with little or no starch taste). The commercial variety Passionis sold and distributed through Monsanto (St. Louis, Mo.), and thecommercial variety Beyond is sold and distributed by Abbott and Cobb,Inc. (Trevose, Pa.), and has served as a standard, primarily in theSoutheast commercial shipping markets. FIG. 13 shows that, in thiscomparison, the variety Passion remained very sweet through the firstfour days following the prime eating stage, and then tapered off in days5, 6 and 7. The variety Beyond was not considered as sweet initially asthe variety Passion, but followed a general pattern of sugar retentionthrough the first four days after the prime eating stage, and then alsotapered off through days 5, 6 and 7. Sweet corn hybrid ACX SS 1082Ysh2-i was similar in sweetness to the Beyond variety in days 1, 2 and 3following the prime eating stage (having a score of about 7, 6 and 5.5on these days, respectively), and then tapered off in days 4, 5, 6 and 7(to scores of about 4, 3, 2 and 2, respectively). The initiation of asomewhat starchy taste was detected in sweet corn hybrid ACX SS 1082Ysh2-i starting on day 3.

FIG. 14 is a line graph that shows organoleptic average pericarptenderness scores for the same sweet corn varieties in the seven dayperiod immediately following the prime eating stage (at a level ofapproximately 75% moisture). It illustrates the comparative organolepticpericarp tenderness levels among these three sweet corn varieties overthis seven day period. The organoleptic tenderness scores ranged from 1(very tough pericarp) to 10 (very tender pericarp). FIG. 14 shows thatthe tenderness level of the Passion variety went from a score of about9.5 on day 1 past the prime eating stage to a score of about 7 on day 7,with the tenderness level holding through the first four days, and thendropping moderately on days 5, 6 and 7. This variety was considered tobe the most tender of the three comparison varieties. The variety Beyondhad a tenderness score of about 6.5 on day 1 past the prime eatingstage, and a score of about 6 on day 2, and then tapered off with scoresof about 5 on day 3, about 4.5 on day 4, and about 4 on days 5, 6 and 7.Thus, this variety was moderately tender in days 1 and 2, and thenbecame substantially less tender starting on day 3 and ending on day 7.The sweet corn hybrid ACX SS 1082Y sh2-i had tenderness scores of about8 on day 1 following the prime eating stage, about 7 on day 2, about 6on day 3, about 5 on day 4, and about 4 (or a little higher) on days 5,6 and 7. Thus, this variety was quite tender on days 1 and 2, slightlyless tender on days 3 and 4, and then moderately tender on days 5, 6 and7.

These data indicate a very beneficial field emergence and vigor benefitto hybrids such as ACX SS 1082Y sh2-i. However, the construction of suchhybrids as described in Examples 6 and 7 indicate that male parent linescontaining but one set of mutant homozygous recessive alleles, forexample Su1Su1Se1Se1 sh2sh2, results in a reduced level of overallsweetness and tenderness in the hybrid combination. The sweetness andpericarp tenderness scores that are presented also suggest that aninsertion into female inbred lines of the mutant sh2-i gene without asequential layering against a genetic background of se1se1 and su1su1,as has been described previously herein, results in less efficacious andsome detrimental effects in connection with overall eating quality(sweetness and pericarp tenderness).

Example 8 Production without using Molecular Markers, and ase1se1/su1su1 Genetic Background, and Testing of Sweet Corn Hybrids ACRSS 4500Y and ACR SS 4501Y sh2-i Female Parents in Combination with MaleParents having Double or Triple Recessive Allelic Combinations

In the experiments that are described in this example, additional sweetcorn sh2-i hybrids were developed using double and triple recessive maleparental genotypes.

Construction of Hybrids ACR SS 4500Y and ACR SS 4501Y

These additional sweet corn hybrids were assembled according to the samebreeding procedures that are described in Examples 1, 2 and 3, with theexceptions that the hybrid lines containing the mutant sh2-i gene intheir genomes were not assembled utilizing molecular markers, aspreviously described herein (Examples 1, 2 and 3), and did notincorporate se1se1/su1su1 alleles.

Two resulting sweet corn sh2-i hybrids of particular interest weredesignated ACR SS 4500Y and ACR SS 4501Y, and were developed usingparental sweet corn inbreds not constructed with su1 and se1backgrounds. These hybrids were developed from sh2-i female conversionparent lines not containing the su1 and se1 genes to test eating qualityin comparison with sh2-i female parental inbreds constructed utilizingthe incorporation of the su1 and se1 genes. It was also consideredimportant to test the effect of using double and triple homozygousrecessive male lines with the sh2-i conversion females lines that werenot constructed utilizing sequential layering over the su1 and se1genes.

These hybrid lines differed in a beneficial manner from other hybridsprepared in this example in performance relative to geographical growingregions and/or market requirements, and their characteristics aredescribed in detail in the Characteristics Table (Hybrid Sweet Cornlines) that is set forth hereinabove prior to Example 1. TheCharacteristics Table (Parental Sweet Corn lines) describes thecharacteristics in detail of the parent sweet corn lines that wereemployed to develop these hybrid lines.

With both hybrid lines ACR SS 4500Y and ACR SS 4501Y, female parentlines were constructed using the mutant sh2-i gene not in combinationwith the su1 and/or se1 genes. The hybrid ACR SS 4500Y line, however,was developed using a triple recessive male parent line, whereas thehybrid ACR SS 4501Y line was developed using a double recessive maleparent line.

Parent female line AC 098Y-i (an sh2-i conversion inbred including themutant sh2-i gene in its genome) is an sh2-i gene isogenic conversionof, and comparison to, a commercially available (from Abbott and Cobb,Inc.) parent female inbred line designated as AC 098Y (not including themutant sh2-i gene in its genome). The only significant geneticdifference between these two inbred female parent lines in this exampleis the presence of the mutant sh2-i gene in inbred female parent line AC098Y-i and the absence of the sh2-i gene in inbred female parent line AC098Y.

Parent female line AC 157Y-i (an sh2-i conversion inbred including themutant sh2-i gene in its genome) is an sh2-i gene isogenic conversionof, and comparison to, an Abbott and Cobb, Inc. parent female linedesignated as AC 157Y (not including the mutant sh2-i gene in itsgenome). The only significant genetic difference between these twoinbred female parent lines in this example is the presence of the mutantsh2-i gene in inbred female parent line AC 157Y-i and the absence of thesh2-i gene in inbred female parent line AC 157Y.

Laboratory Warm and Cold Germination Testing and Organoleptic Taste andPericarp Tenderness Testing

Table 10 below provides data resulting from actual comparisons oflaboratory warm and cold germination data, as well as organoleptic tasteand pericarp tenderness tests, for the sweet corn inbred female parentlines AC 098Y-i, AC 098Y, AC 157Y-i, and AC 157Y. Germination scores aremeans of three replications of 100 kernels each. Laboratory scores notfollowed by the same number are significantly different at the 0.05probability level via the Duncan's New Multiple Range Test. Theorganoleptic tests regarding sweetness (sugar content) and pericarptenderness are described previously herein.

Table 10 shows that, in both cases in which the sh2-i mutant gene wasadded to the sweet corn genomes (inbred female parent lines AC 098Y-iand AC 157Y-i), the isogenic inbred comparison warm and cold laboratoryscores were significantly elevated in comparison with the inbred femaleparent lines not including the sh2-i mutant gene in their genomes (AC098Y and AC 157Y), which is indicative of an enhanced field emergenceand vigor. Table 10 also shows that these two sweet corn inbreds linesincluding the mutant sh2-i gene did not retain their sweetness quite aslong (having scores of 2 and 2, respectively, on Day 7 in the seven dayperiod immediately following the prime eating stage) as the two sweetcorn inbred lines that did not include this gene in their genomes(inbred AC 098Y and AC 157Y) (having scores of 5 and 4 on Day 7), andhad pericarps that did not retain their tenderness quite as long (havingscores of 1 and 2, respectively, on Day 7 in the seven day periodimmediately following the prime eating stage) as the two sweet corninbreds that did not include this gene in their genomes (having scoresof 3 and 4, respectively, on Day 7). The sweetness and pericarptenderness scores that are present in Table 10 suggest that an insertioninto inbred lines of the mutant sh2-i gene without a sequential layeringagainst a genetic background of se1se1 and su1su1, as has been describedpreviously herein, results in less efficacious and some detrimentaleffects in connection with overall eating quality (sweetness andpericarp tenderness) in comparison with an insertion into the sameinbred lines of the mutant sh2-i gene with. a sequential layeringagainst a genetic background of se1se1 and su1su1. They all, however,very advantageously exhibit an enhanced field emergence and vigor.

In view of the above, it is considered to be very desirable to assembleand direct the construction of mutant sh2-i gene materials in the mannerthat has been described previously herein in order to obtain maximumbenefits desired by plant growers and consumers (i.e., using molecularmarkers and an se1se1 and su1su1 genetic background).

TABLE 10 Isogenic Comparisons of Sweet Corn Inbreds Including, or notIncluding, the sh2-i Gene Organoleptic Sweetness Organoleptic PericarpWarm Cold Score Tenderness Score Inbreds Germination Germination Day 1Day 4 Day 7 Day 1 Day 4 Day 7 AC 098Y-i 99a 90a 8 5 2 7 3 1 (Containssh2-i Gene) AC 098Y 98a 82b 8 6 5 8 5 3 (Does not Contain sh2-i Gene) AC157Y-i 98a 94a 8 2 2 7 5 2 (Contains sh2-i Gene) AC 157Y 94a 71c 8 5 4 76 4 (Does not Contain sh2-i Gene)

Additional Deposits Deposits made with the American Type CultureCollection (ATCC) in Connection with Examples 6-8

The seed deposits discussed below, which relate to Examples 6-8, havebeen made with the American Type Culture Collection (ATCC) (Manassas,Va., USA) by Applicant Bryant J. Long on behalf of assignee Abbott &Cobb, Inc. (Wellington, Fla.) for the Zea mays, sweet corn hybridsidentified below having desired specific allelic combinations, whicheach include the sh2-i gene in their genomes, and the corresponding Zeamays, sweet corn parent lines that were employed to develop these sweetcorn hybrids. In the tables shown below, in connection with the sugary(su1), sugary enhancer-1 (se1) and shrunken-2 (sh2) genes, thedesignation “S” indicates that the male parent line included a singlerecessive allelic combination of these three genes, the designation “D”indicates that the male parent line included a double recessive alleliccombination of these three genes, and the designation “T” indicates thatthe male parent line included a triple recessive allelic combination ofthese three genes.

Example Deposit Allelic Hybrid Parent Lines No. Made Combination ACX SSAC 157Y-i (Female) × 7 Yes* S 1082Y AC 144Y (Male) sh2-i ACR SS AC098Y-i (Female) × 8 Yes* T 4500Y AC 233Y (Male) ACR SS AC 157Y-i(Female) × 8 Yes* D 4501Y AC 241Y (Male) *All three lines (two parentsand hybrid) have been deposited.

ATCC Patent Identification Reference Deposit Used by Depositor/InventorDesignation Quantity Received (1) Sweet Corn, Zea mays: PTA-11467 100packets/25 seeds ACX SS 1082Y sh2-i in each packet (2) Sweet Corn, Zeamays: PTA-11470 100 packets/25 seeds AC 157Y-i in each packet (3) SweetCorn, Zea mays: PTA-11469 100 packets/25 seeds AC 144Y in each packet(4) Sweet Corn, Zea mays: PTA-11472 100 packets/25 seeds ACR SS 4500Y ineach packet (5) Sweet Corn, Zea mays: PTA-11471 100 packets/25 seeds AC098Y-i in each packet (6) Sweet Corn, Zea mays: PTA-11465 100 packets/25seeds AC 233Y in each packet (7) Sweet Corn, Zea mays: PTA-11468 100packets/25 seeds ACR SS 4501Y in each packet (8) Sweet Corn, Zea mays:PTA-11466 100 packets/25 seeds AC 241Y in each packet

Hybrid Lines

Seeds of sweet corn, Zea mays, hybrids ACX SS 1082Y sh2-i, ACR SS 4500Yand ACR SS 4501Y (100 packets for each of these three hybrids, with 25seeds in each packet) were deposited on behalf of Abbott & Cobb; Inc.with the American Type Culture Collection (ATCC) (10801 UniversityBoulevard, Manassas, Va., 20110-2209, United States of America) underThe Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms on Nov. 11, 2010, and were given ATCC Patent DepositDesignations PTA-11467, PTA-11472 and PTA-11468, respectively, by theATCC. The deposited seeds were tested on Nov. 22, 2010 by the ATCC and,on that date, they were viable. The depositor waste Abbott & Cobb, Inc.,11460 Fortune Circle, Wellington, Fla., 33414, United States of America.

Parent Lines

Seeds of the sweet corn, Zea mays, female and male parent lines thatwere employed to develop sweet corn, Zea mays, hybrid ACX SS 1082Y sh2-i(100 packets, with 25 seeds in each packet), designated AC 157Y-i(female) and AC 144Y (male), were deposited on behalf of Abbott & Cobb,Inc. with the American Type Culture Collection (ATCC) (10801 UniversityBoulevard, Manassas, Va., 20110-2209, USA) under The Budapest Treaty onthe International Recognition of the Deposit of Microorganisms on Nov.11, 2010, and were given ATCC Patent Deposit Designations PTA-11470 andPTA-11469 by the ATCC, respectively. The deposited seeds were tested onNov. 22, 2010 by the ATCC and, on that date, they were viable.

Seeds of the sweet corn, Zea mays, female and male parent lines thatwere employed to develop sweet corn, Zea mays, hybrid ACR SS 4500Y (100packets, with 25 seeds in each packet), designated AC 098Y-i (female)and AC 233Y (male), were deposited on behalf of Abbott & Cobb, Inc. withthe American Type Culture Collection (ATCC) (10801 University.Boulevard, Manassas, Va., 20110-2209, USA) under The Budapest Treaty onthe International Recognition of the Deposit of Microorganisms on Nov.11, 2010, and were given ATCC Patent Deposit Designations PTA-11471 andPTA-11465 by the ATCC, respectively. The deposited seeds were tested onNov. 22, 2010 by the ATCC and, on that date, they were/are viable.

Seeds of the sweet corn, Zea mays, female and male parent lines thatwere employed to develop sweet corn, Zea mays, hybrid ACR SS 4501Y (100packets, with 25 seeds in each packet), designated AC 157Y-i (female)and AC 241Y (male), were deposited on behalf of Abbott & Cobb, Inc. withthe American Type Culture Collection (ATCC) (10801 University Boulevard,Manassas, Va., 20110-2209, USA) under The Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms on Nov. 11,2010, and were given ATCC Patent Deposit Designations PTA-11470 andPTA-11466 by the ATCC, respectively. The deposited seeds were tested onNov. 22, 2010 by the ATCC and, on that date, they were viable.

Certificates of Deposit have been received from the American TypeCulture Collection (ATCC) for each of the deposits that are describedabove.

Beneficial Traits and Characteristics of Hybrids including the sh2-iGene

The data that are present in Tables 6 and 7 (Examples 7 and 8—inbredparent sweet corn lines) and Tables 8 and 9 (Example 7—hybrid sweet cornlines), and in the other tables and figures described and illustratedherein, clearly demonstrate the greatly enhanced warm and coldlaboratory and field soil germination, seedling vigor and plant strengthof individual inbred parent and hybrid sweet corn seeds and varietiescontaining the sh2-i gene in both various double and triple alleliccombinations in comparison with the same or similar inbred parent andhybrid sweet corn seeds and varieties (in both double and triple alleliccombinations) that do not contain the sh2-i gene. These multiple veryimportant and very significant advantageous sweet corn seed and planttraits and growing benefits are extremely beneficial and helpful forsweet corn growers and producers worldwide, and in many different typesand climates of various geographical areas and regions employed forgrowing sweet corn worldwide, for example, in Northern areas or regionsof various countries and/or states, such as the United States, wheresweet corn growing temperatures and/or climates are often significantlycooler in comparison with Southern regions, where growing temperaturesand climates are often significantly hotter, and where maintaining auniform, consistent sweet corn stand establishment is often difficultfor sweet corn producers. Sweetness and pericarp tenderness expectationsof these sweet corn varieties are considered to be excellent,particularly in situations in which sweet corn products can be deliveredto consumer markets within an approximately 48 hour timeframe.

In conclusion, the sweet corn seeds and varieties that are discussed andillustrated herein (including an sh2-i gene) very advantageously havenumerous very advantageous and very beneficial traits in comparison withother known or other sweet corn seeds and varieties (not including thesh2-i gene): (i) an enhanced laboratory warm germination from soil; (ii)an enhanced laboratory cold germination from soil; (iii) an enhancedgermination from soil generally in the field; (iv) an enhanced seedlingvigor; (v) an enhanced commercial seed production yield; (vi) anenhanced plant strength; (vii) an extended shelf life; (viii) a sweettaste while also maintaining beneficial traits (i)-(vii) and (ix); and(ix) a tender pericarp while also maintaining beneficial traits(i)-(viii). Many of these sweet corn seeds and varieties additionallyand very advantageously exhibit disease resistance, for example, tonorthern corn leaf blight and/or to multiple races of common rust.

Further, the hybrid seeds produced according to the methods of theinvention generally have a markedly different appearance from othertypes of corn seed. From a visual observation of the seeds, one can seethe difference in their appearance from other corn seed. They do notappear to have a shrunken appearance, store starches, are generallytriangular in shape.

Strategy Employed and Failure of Others

Although the shrunken-2i (sh2-i) and sugary (su1) genes are known, noone to date has been able to successfully combine these genes to producesweet corn hybrids expressing enhanced seedling germination and vigoralong with the retention of desirable consumer and/or grower traits.There has been a long-felt, but unresolved industry need for costeffective, reliable hybrids expressing an enhanced seedling germination,vigor, and plant development (like conventional sh2-i sweet corn), butwith an enhanced holding ability of high sugar and tender pericarplevels both on and off the plant. Improved growth and tastecharacteristics of sweet corn would confer a significant advancement inthe commercial production of these plants.

Various commercial plant breeders and companies, such as Syngenta SeedsInc., which have at least “ordinary skill in the art,” have attemptedand failed to successfully combine traits to achieve sweet cornvarieties utilizing sh2-i that is pleasing to both growers andconsumers. While Syngenta has one commercial variety (named “Brighton”)that includes the sh2-i allele in its genome, this product has not beencommercially successful because the eating quality and, specifically,its holding ability are not desirable to consumers.

At least one other company employing plant breeders that have at least“ordinary skill in the art” also procured the sh2-i gene in a donorsource from The University of Florida (as the inventor did). However, incontrast with the present inventor, this company was unable tosuccessfully use the sh2-i gene to produce desirable sweet corn parentaland hybrid lines. In contrast, the present inventor was able toincorporate the sh2-i gene very successfully into plant genomes in amanner to produce parental and hybrid line sweet corn seeds, plantmaterials and plants having multiple enhanced traits, such as those thatare discussed hereinabove, including an enhanced growth and vigor ofseeds and plants (a trait provided by the homozygous recessive sh2-iallele). The present inventor was able to accomplish this after asignificant amount of experimentation and testing and a formulation andinstitution of a strategy to use: (i) the homozygous recessive sh2-iallele in a female parent line, and only in a female parent line (i.e.,not in a male parent line), as is discussed in the “Examples” sectionherein (providing an enhanced growth and vigor to the seeds and plants);and (ii) homozygous recessive forms of different sweet corn allelesproviding different traits to hybrids developed therefrom, such ashaving a sweet taste, a tender pericarp and a long shelf life(characteristics provided by other sweet corn alleles, which the presentinventor used in hybrids in homozygous recessive forms along with thesh2-i gene).

Further, when the University of Florida provided a “donor source” of thesh2-i allele to its various licensees (including the present inventorand at least one unrelated company employing plant breeders), itprovided the sh2-i allele in a form that is homozygous recessive for thesh2-i allele, but that was homozygous dominant (Su1Su1 Se1Se1) relativeto the sugary-1 (su1) and sugary enhancer-1 (se1) mutant endospermalleles (i.e., it had homozygous dominant forms of these two alleles).Because the sh2-i allele was homozygous recessive (sh2-i sh2-i), itprovided enhanced seedling performances that are associated with themutant sh2-i gene (which is recessive and, thus, must be homozygousrecessive in order to have this trait expressed). Thus, the donor sourceof the sh2-i allele had the genotype Su1Su1 Se1Se1 sh2-i sh2-i. To date,none of the companies that have licensed the sh2-i allele from theUniversity of Florida apart from the assignee of the present inventionhave been able to develop a commercially acceptable sweet corn hybridvariety containing the sh2-i gene for the marketplace.

Using molecular markers, the present inventor was able to determine thegenotype of the donor source of the sh2-i allele. As is described in theExamples section hereof, the inventor was able to figure out that thehomozygous recessive sh2-i sh2-i allele (producing the desirableseedling vigor trait, since both alleles are recessive) should bepresent against a genetic background of: (i) su1su1 se1se1 (a doublerecessive homozygous allelic combination) in a female parent line toproduce a genotype of su1su1 se1se1 sh2-i sh2-i. (a triple homozygousrecessive allelic combination, so that the individual beneficial traitsof each of the three types of alleles were each expressed, and notmasked by an inclusion of any dominant alleles); or (ii) su1su1 (singlerecessive homozygous alleles) in a female parent line to produce agenotype of su1su1 sh2-i sh2-i. (a double homozygous recessive alleliccombination, so that the individual beneficial traits of both of the twotypes of alleles were each expressed, and not masked by an inclusion ofany dominant alleles).

The inventor also performed various organoleptic taste and pericarptenderness testing and discovered that unacceptable starch synthesis andbuildup following the peak eating stage disallowed the process ofassembling hybrids based upon the use of two sh2-i parental corn lines(i.e., with both the male and female parent lines including sh2-ialleles). The inventor subsequently determined that the best alternativewas to be a generation of sweet corn hybrids comprised of an sh2-ifemale sweet corn line by a high-quality conventional male parental cornline.

Thus, although the University of Florida had possession of the sweetcorn sh2-i allele, it did not have it (or use it) in a genotype that washomozygous recessive for all of the alleles affecting traits of sweetcorn, which was determined by the present inventor to be one of the mostsignificant aspects of making it work when used in a parent line toproduce hybrid seeds, plants and plant materials having an enhancedseedling vigor and growth when crossing with another parent line havingone or more different beneficial traits, such as enhanced sweetness,pericarp tenderness and/or shelf life. The inventor determined thatanother very important aspect was to include the sh2-i allele (in arecessive homozygous form) only in the female parent line. A furtherimportant aspect was determined to cross such a female parent line withanother parent line including one or more alleles, or alleliccombinations, that were all in a homozygous recessive form (i.e. triplehomozygous recessive, double homozygous recessive, single homozygousrecessive, or the like). In such a combination of parent lines, nodominant traits could result and mask any of the desirable recessivetraits, and the recessive genotype would result in a phenotype includingdesired beneficial traits provided by recessive alleles. (In contrast,when either or both of the parent lines that are crossed to producehybrids include alleles in a heterozygous form (i.e., including onedominant allele for one or more types of allele), or in a homozygousdominant form (i.e., including two dominant alleles for one or moretypes of alleles), the resulting phenotype of some or all of theresulting hybrid sweet corn seeds would result from the dominant alleles(i.e., not providing a beneficial trait to sweet corn hybrids developedtherefrom), not from the recessive alleles (providing a beneficial traitwhen present in a homozygous recessive form). The dominant alleles havean effect of masking the traits of the recessive alleles, which providethe beneficial phenotypic traits.

Others who licensed the sh2-i gene from the University of Florida hadthe same problem that the present inventor initially had with the donorsource of sh2-i allele, but were not able to figure out how to solvethis problem, or to produce hybrid sweet corn seeds, plant materials orplants having the beneficial trait of an enhanced seedling vigor whenusing the licensed sh2-i allele. In contrast, the present inventorsolved this very difficult problem using molecular markers and a varietyof different tests (organoleptic taste and pericarp tenderness testing,laboratory warm and cold germination testing, field testing, seedlingvigor testing and the like), as is described in the “Examples” sectionherein. The present inventor was able to use the sh2-i allele in aparent line in a homozygous recessive form and cross it with anotherparent line to produce seeds, plant materials and plants having: (i) asweet taste (due to one or more different alleles in a homozygousrecessive form); (ii) a long shelf life (due to one or more differentalleles in a homozygous recessive form); and (iii) an enhanced seedlingvigor (due to the sh2-i allele in a homozygous recessive form used in afemale parent line), as well as the other beneficial traits that aredescribed herein.

Further, all of the entities that licensed the sh2-i allele from TheUniversity of Florida would not expect for a licensed allele to have theproblems discussed above.

While the present invention has been described herein with specificity,and with reference to certain preferred embodiments thereof, those ofordinary skill in the art will recognize numerous variations,modifications and substitutions of that which has been described whichcan be made, and which are within the scope and spirit of the invention.It is intended that all of these modifications and variations be withinthe scope of the present invention as it is described and claimedherein, and that the invention be limited only by the scope of theclaims which follow, and that such claims be interpreted as broadly asis reasonable.

Throughout this document, various books, patents, journal articles, websites and other publications have been cited. The entireties of each ofthese books, patents, journal articles, web sites and other publicationsare hereby incorporated by reference herein.

What is claimed is:
 1. A method for producing a hybrid maize plant,plant material or seed that has enhanced vigor in comparison with aconventional mutant shrunken-2 hybrid maize plant, plant material orseed and one or more additional desirable traits, including an enhancedability to retain sugar over a period of time following a prime eatingstage thereof, in comparison with a conventional mutant sugary-1 orshrunken-2i hybrid maize plant, plant material or seed, comprising thefollowing steps: (a) identifying an inbred maize plant line thatincludes one or more desired mutant endosperm alleles in its genome,singly or in combination, optionally using one or more molecularmarkers, wherein the mutant endosperm alleles are sugary-1 (su1), sugaryenhancer-1 (se1) or shrunken-2 (sh2), or any combinations thereof; (b)constructing one or more female near isogenic maize plant lines having adesired genotype for use as a genetic background in a combination with afemale parental maize plant line that includes a shrunken-2i (sh2-i)mutant endosperm allele in its genome; (c) incorporating a shrunken-2imutant endosperm allele into the genome of the female near isogenicmaize plant line having the desired genetic background of step (b),optionally using one or more molecular markers, wherein the desiredgenetic background is Su1Su1 se1se1, Su1Su1 se1se1 sh2sh2, Se1Se1su1su1, Se1Se1 su1su1 sh2sh2, su1su1 se1se1, su1su1 sh2sh2, se1se1sh2sh2, su1su1 se1se1 sh2sh2, or another desired genetic backgroundincluding in homozygous recessive condition the sugary-1 and/or sugaryenhancer-1 mutant endosperm alleles; (d) selecting a female convertednear isogenic maize plant line of step (c) having a shrunken-2i mutantendosperm allele incorporated into the genetic background of step (c),wherein the female converted near isogenic maize plant line includes anendosperm allelic combination of Su1Su1 se1se1 sh2-i sh2-i, Se1Se1su1su1 sh2-i sh2-i, su1su1 se1se1 sh2-i sh2-i, su1su1 sh2-i sh2-i,se1se1 sh2-i sh2-i, or another desirable endosperm allelic combinationof the shrunken-2i mutant endosperm allele in homozygous conditiontogether with either or both of the sugary-1 or sugary enhancer-1 mutantendosperm alleles each in homozygous condition, in its genome; (e)crossing the selected female maize converted near isogenic plant line ofstep (d) with a male maize plant line that does not include ashrunken-2i mutant endosperm allele in its genome, and that includes adouble homozygous recessive endosperm allelic construction in itsgenome, wherein such double homozygous recessive endosperm allelicconstruction is Su1Su1 se1se1 sh2sh2, su1su1 Se1Se1 sh2sh2, su1su1sh2sh2, se1se1 sh2sh2, or another double homozygous recessive endospermallelic combination including either the sugary-1 or sugary enhancer-1mutant endosperm alleles together with the shrunken-2 mutant endospermallele, that can provide a hybrid maize plant with a high or enhancedeating quality; to produce a hybrid maize plant comprising the allelicconstruction sh2 sh2-i at the shrunken-2 locus and homozygous recessivealleles at either or both of the su1 and se1 loci, said hybrid maizeplant having one or more desired grower traits, consumer traits, or bothtraits; (f) optionally, examining a physical appearance of seeds orkernels produced by the maize plants of step (d), step (e), or both step(d) and step (e), for characteristics including smoothness, fullness orrelative weight, or a combination thereof; (g) optionally, conductingone or more warm, cold, or both warm and cold, laboratory, field, orlaboratory and field, germinations of seeds or kernels produced by themaize plants of step (d), step (e), or both step (d) and step (e), toverify that such seeds or kernels have one or more desired consumer orgrower traits, or a combination thereof; and (h) optionally, conductingone or more organoleptic taste, pericarp tenderness or other tests onmaize plants, or plant parts, or a combination thereof, that are grownfrom seeds or kernels produced by the maize plants of step (d), step(e), or both step (d) and step (e), to determine their taste, pericarptenderness or other organoleptic characteristics, or a combinationthereof; wherein the hybrid maize plant, plant material or seed has anenhanced vigor in comparison with a conventional mutant shrunken-2hybrid maize plant, plant material or seed; and wherein the hybrid maizeplant, plant material or seed has an enhanced ability to retain sugarover a period of time following a prime eating stage thereof incomparison with a conventional mutant sugary-1 or shrunken-2i hybridmaize plant, plant material or seed.
 2. A method of claim 1, wherein theselected female maize converted near isogenic plant line of step (d)includes the allelic combination su1su1 se1se1 sh2-i sh2-i, and the malemaize plant line of step (e) includes the allelic combination se1se1sh2sh2, su1su1 sh2sh2 or su1su1 Se1Se1 sh2sh2.
 3. A method of claim 2,wherein one or more molecular markers are employed to incorporate theshrunken-2i mutant endosperm allele into the genome of the female maizenear isogenic plant line of step (c).
 4. A method of claim 1, whereinmolecular markers are employed in step (a) to identify the inbred maizeplant line that includes one or more desired mutant endosperm alleles inits genome.
 5. A method of claim 1, wherein the female near isogenicmaize plant lines of step (b) include one of the following alleliccombinations: (i) su1su1 Se1Se1 sh2sh2; (ii) Su1Su1 se1se1 sh2sh2; (iii)su1su1 se1se1 sh2sh2; (iv) su1su1 se1se1; or (v) su1su1 sh2sh2.
 6. Amethod of claim 5, wherein the allelic combination is su1su1 se1se1sh2sh2 or su1su1 se1se1.
 7. A method of claim 5, wherein one or moremolecular markers are employed in step (c) to incorporate theshrunken-2i mutant endosperm allele into the genome of the female maizenear isogenic plant line having a desired genetic background.
 8. Amethod of claim 6, wherein one or more molecular markers are employed instep (c) to incorporate the shrunken-2i mutant endosperm allele into thegenome of the female maize near isogenic plant line having a desiredgenetic background.
 9. A method of claim 7, wherein the desired geneticbackground of step (c) is su1su1 se1se1 sh2sh2, su1su1 se1se1, Su1Su1se1se1 sh2sh2, or Su1Su1 se1se1.
 10. A method of claim 8, wherein thedesired genetic background of step (c) is su1su1 se1se1 sh2sh2, su1su1se1se1, Su1Su1 se1se1 sh2sh2, or Su1Su1 se1se1.
 11. A method of claim 5,wherein the female maize converted near isogenic plant line of step (d)is crossed with a male maize plant line including a double homozygousrecessive endosperm allelic combination of su1 su1 sh2sh2, se1se1 sh2sh2or su1su1 Se1Se1 sh2sh2 in its genome.
 12. A method of claim 6, whereinthe female maize converted near isogenic plant line of step (d) iscrossed with a male maize plant line including a double homozygousrecessive endosperm allelic combination of su1su1 sh2sh2, se1se1 sh2sh2or su1su1 Se1Se1 sh2sh2 in its genome.
 13. A method of claim 7, whereinthe female maize converted near isogenic plant line of step (d) iscrossed with a male maize plant line including a double homozygousrecessive endosperm allelic combination of su1su1 sh2sh2, se1se1 sh2sh2or su1su1 Se1Se1 sh2sh2 in its genome.
 14. A method of claim 8, whereinthe female maize converted near isogenic plant line of step (d) iscrossed with a male maize plant line including a double homozygousrecessive endosperm allelic combination of su1su1 sh2sh2, se1se1 sh2sh2or su1su1 Se1Se1 sh2sh2 in its genome.
 15. A method of claim 9, whereinthe female maize converted near isogenic plant line of step (d) iscrossed with a male maize plant line including a double homozygousrecessive endosperm allelic combination of su1su1 sh2sh2, se1se1 sh2sh2or su1su1 Se1Se1 sh2sh2 in its genome.
 16. A method of claim 10, whereinthe female maize converted near isogenic plant line of step (d) iscrossed with a male maize plant line including a double homozygousrecessive endosperm allelic combination of su1su1 sh2sh2, se1se1 sh2sh2or su1su1 Se1Se1 sh2sh2 in its genome.
 17. A method of claim 11, whereinthe double homozygous recessive endosperm allelic combination in themale plant is su1su1 sh2sh2 or su1su1 Se1Se1 sh2sh2.
 18. A method ofclaim 12, wherein the double homozygous recessive endosperm alleliccombination in the male plant is su1su1 sh2sh2 or su1su1 Se1Se1 sh2sh2.19. A method of claim 13, wherein the double homozygous recessiveendosperm allelic combination in the male plant is su1su1 sh2sh2 orsu1su1 Se1Se1 sh2sh2.
 20. A method of claim 14, wherein the doublehomozygous recessive endosperm allelic combination in the male plant issu1su1 sh2sh2 or su1su1 Se1Se1 sh2sh2.
 21. A method of claim 15, whereinthe double homozygous recessive endosperm allelic combination in themale plant is su1su1 sh2sh2 or su1su1 Se1Se1 sh2sh2.
 22. A method ofclaim 16, wherein the double homozygous recessive endosperm alleliccombination in the male plant is su1su1 sh2sh2 or su1su1 Se1Se1 sh2sh2.23. A method of claim 17, wherein the double homozygous recessiveendosperm allelic combination in the male plant is su1su1 sh2sh2.
 24. Amethod of claim 18, wherein the double homozygous recessive endospermallelic combination in the male plant is su1su1 sh2sh2.
 25. A method ofclaim 19, wherein the double homozygous recessive endosperm alleliccombination in the male plant is su1su1 sh2sh2.
 26. A method of claim20, wherein the double homozygous recessive endosperm alleliccombination in the male plant is su1su1 sh2sh2.
 27. A method of claim21, wherein the double homozygous recessive endosperm alleliccombination in the male plant is su1su1 sh2sh2.
 28. A method of claim22, wherein the double homozygous recessive endosperm alleliccombination in the male plant is su1su1 sh2sh2.
 29. A method of claim 5,wherein a physical appearance of seeds or kernels produced by the plantsof step (d), step (e), or both step (d) and step (e), is examined forcharacteristics including smoothness, fullness or relative weight, or acombination thereof.
 30. A method of claim 6, wherein a physicalappearance of seeds or kernels produced by the plants of step (d), step(e), or both step (d) and step (e), is examined for characteristicsincluding smoothness, fullness or relative weight, or a combinationthereof.
 31. A method of claim 5, wherein warm, cold, or both warm andcold, laboratory, field, or both laboratory and field, germinations ofseeds or kernels produced by the plants of step (d), step (e), or bothstep (d) and step (e), are conducted to verify that such seeds orkernels have an enhanced seedling germination, an enhanced seedlingemergence, or an enhanced plant growth, or a combination thereof, eachin comparison with a conventional mutant shrunken-2 maize plant, plantmaterial or seed; or an enhanced ability to retain sugar over a periodof time following a prime eating stage thereof, in comparison with aconventional mutant sugary-1 or shrunken-2i maize plant, plant material,or seed; or both.
 32. A method of claim 6, wherein warm, cold, or bothwarm and cold, laboratory, field, or both laboratory and field,germinations of seeds or kernels produced by the plants of step (d),step (e), or both step (d) and step (e), are conducted to verify thatsuch seeds or kernels have an enhanced seedling germination, an enhancedseedling emergence, or an enhanced plant growth, or a combinationthereof, each in comparison with a conventional mutant shrunken-2 maizeplant, plant material or seed; or an enhanced ability to retain sugarover a period of time following a prime eating stage thereof, incomparison with a conventional mutant sugary-1 or shrunken-2i maizeplant, plant material, or seed; or both.
 33. A method of claim 5,wherein one or more organoleptic taste, pericarp tenderness, or othertests on plants, or plant parts, that are grown from seeds or kernelsproduced by the plants of step (d), step (e), or both step (d) and step(e), or any combination thereof, are conducted to determine their taste,pericarp tenderness or other organoleptic characteristics, or anycombination thereof.
 34. A method of claim 6, wherein one or moreorganoleptic taste, pericarp tenderness, or other tests on plants, orplant parts, that are grown from seeds or kernels produced by the plantsof step (d), step (e), or both step (d) and step (e), or any combinationthereof, are conducted to determine their taste, pericarp tenderness orother organoleptic characteristics, or any combination thereof.
 35. Amethod of claim 1, wherein the method produces an edible maize plant,plant material, seed or kernel.
 36. A method of claim 35, wherein themethod produces a sweet corn plant, plant material, seed or kernel. 37.A method of claim 1, wherein the hybrid maize plant, plant material orseed has an enhanced ability to retain sugar in days 1-7, days 1-14 ordays 7-14 immediately following a prime eating stage thereof incomparison with a conventional mutant sugary-1 or shrunken-2i hybridplant, plant material or seed.
 38. A method of claim 35, wherein themaize plant, plant material, seed or kernel is classified as Zea maysvar. rugosa.
 39. A method of claim 3, wherein the hybrid maize plant,plant material or seed is classified as Zea mays var. rugosa, and has anenhanced ability to retain sugar in days 1-7, days 1-14 or days 7-14immediately following a prime eating stage thereof in comparison with aconventional mutant sugary-1 or shrunken-2i hybrid plant, plant materialor seed.
 40. A method of claim 1, wherein the hybrid maize plant, plantmaterial or seed has an enhanced ability to retain sugar in days 7-14immediately following a prime eating stage thereof in comparison with aconventional mutant sugary-1 or shrunken-2i hybrid plant, plant materialor seed.
 41. A method of claim 37, wherein the hybrid maize plant, plantmaterial or seed has an enhanced ability to retain sugar in days 7-14immediately following a prime eating stage thereof in comparison with aconventional mutant sugary-1 or shrunken-2i hybrid plant, plant materialor seed.
 42. A method for producing a hybrid maize plant, plantmaterial, or seed comprising: crossing an inbred maize plant line thatis homozygous recessive for the shrunken-2i mutant endosperm allele andalso homozygous recessive for one or both of the su1 or se1 mutantendosperm alleles as the female maize parent plant line; with a malemaize parent plant line that does not include a shrunken-2i allele inits genome, and that is homozygous recessive for the sh2 mutantendosperm allele and one or both of the su1 or se1 mutant endospermalleles; to provide a hybrid maize plant, plant material or seed with ahigh or an enhanced eating quality; wherein the hybrid maize plant,plant material or seed has an enhanced ability to retain sugar over aperiod of time following a prime eating stage thereof in comparison witha conventional mutant sugary-1 or shrunken-2i hybrid maize plant, plantmaterial or seed.
 43. A method of claim 42, wherein the method producesSweet Corn Hybrid ACX SS 7501Y, representative seeds of which weredeposited as ATCC Accession No. PTA-10507; Sweet Corn Hybrid ACX SS7078Y, representative seeds of which were deposited as ATCC AccessionNo. PTA-10506; or Sweet Corn Hybrid ACX SS 7403RY, representative seedsof which were deposited as ATCC Accession No. PTA-10508.
 44. A methodfor producing a hybrid maize plant, plant material or seed that hasenhanced vigor in comparison with a conventional mutant shrunken-2hybrid maize plant, plant material or seed and one or more additionaldesirable traits, including an enhanced ability to retain sugar over aperiod of time following a prime eating stage thereof, in comparisonwith a conventional mutant sugary-1 or shrunken-2i hybrid maize plant,plant material or seed, consisting of the following steps: (a)identifying an inbred maize plant line that includes one or more desiredmutant endosperm alleles in its genome, singly or in combination,optionally using one or more molecular markers, wherein the mutantendosperm alleles are sugary-1 (su1), sugary enhancer-1 (se 1) orshrunken-2 (sh2), or any combinations thereof; (b) constructing one ormore female near isogenic maize plant lines having a desired genotypefor use as a genetic background in a combination with a female parentalmaize plant line that includes a shrunken-2i (sh2-i) mutant endospermallele in its genome; (c) incorporating a shrunken-2i mutant endospermallele into the genome of the female near isogenic maize plant linehaving the desired genetic background of step (b), optionally using oneor more molecular markers, wherein the desired genetic background isSu1Su1 se1se1, Su1Su1 se1se1 sh2sh2, Se1Se1 su1su1, Se1Se1 su1su1sh2sh2, su1su1 se1se1, su1su1 sh2sh2, se1se1 sh2sh2, su1su1 se1se1sh2sh2, or another desired genetic background including in homozygousrecessive condition the sugary-1 and/or sugary enhancer-1 mutantendosperm alleles; (d) selecting a female converted near isogenic maizeplant line of step (c) having a shrunken-2i mutant endosperm alleleincorporated into the genetic background of step (c), wherein the femaleconverted near isogenic maize plant line includes an endosperm alleliccombination of Su1Su1 se1se1 sh2-i sh2-i, Se1Se1 su1su1 sh2-i sh2-i,su1su1 se1se1 sh2-i sh2-i, su1su1 sh2-i sh2-i, se1se1 sh2-i sh2-i, oranother desirable endosperm allelic combination of the shrunken-2imutant endosperm allele in homozygous condition together with either orboth of the sugary-1 or sugary enhancer-1 mutant endosperm alleles eachin homozygous condition, in its genome; (e) crossing the selected femalemaize converted near isogenic plant line of step (d) with a male maizeplant line that does not include a shrunken-2i mutant endosperm allelein its genome, and that includes a double homozygous recessive endospermallelic construction in its genome, wherein such double homozygousrecessive endosperm allelic construction is Su1Su1 se1se1 sh2sh2, su1su1Se1Se1 sh2sh2, su1su1 sh2sh2, se1se1 sh2sh2, or another doublehomozygous recessive endosperm allelic combination including either thesugary-1 or sugary enhancer-1 mutant endosperm alleles together with theshrunken-2 mutant endosperm allele, that can provide a hybrid maizeplant with a high or enhanced eating quality; to produce a hybrid maizeplant comprising the allelic construction sh2 sh2-i at the shrunken-2locus and homozygous recessive alleles at either or both of the su 1 andse 1 loci, said hybrid maize plant having one or more desired growertraits, consumer traits, or both traits; (f) optionally, examining aphysical appearance of seeds or kernels produced by the maize plants ofstep (d), step (e), or both step (d) and step (e), for characteristicsincluding smoothness, fullness or relative weight, or a combinationthereof; (g) optionally, conducting one or more warm, cold, or both warmand cold, laboratory, field, or laboratory and field, germinations ofseeds or kernels produced by the maize plants of step (d), step (e), orboth step (d) and step (e), to verify that such seeds or kernels haveone or more desired consumer or grower traits, or a combination thereof;and (h) optionally, conducting one or more organoleptic taste, pericarptenderness or other tests on maize plants, or plant parts, or acombination thereof, that are grown from seeds or kernels produced bythe maize plants of step (d), step (e), or both step (d) and step (e),to determine their taste, pericarp tenderness or other organolepticcharacteristics, or a combination thereof; wherein the hybrid maizeplant, plant material or seed has an enhanced vigor in comparison with aconventional mutant shrunken-2 hybrid maize plant, plant material orseed; and wherein the hybrid maize plant, plant material or seed has anenhanced ability to retain sugar over a period of time following a primeeating stage thereof in comparison with a conventional mutant sugary-1or shrunken-2i hybrid maize plant, plant material or seed.
 45. A methodof claim 42, wherein the female maize parent plant line includes theallelic combination su1su1 se1se1 sh2-i sh2-i, and the male maize parentplant line includes the allelic combination se1se1 sh2sh2, su1su1 sh2sh2or su1su1 Se1Se1 sh2sh2.
 46. A method of claim 45, wherein one or moremolecular markers are employed to incorporate the shrunken-2i mutantendosperm allele into the genome of the female maize parent plant line.47. A method of claim 42, wherein the female maize parent plant lineincludes homozygous recessive sugary-1 mutant endosperm alleles.
 48. Amethod of claim 42, wherein the female maize parent plant line includeshomozygous recessive sugary enhancer-1 mutant endosperm alleles.
 49. Amethod of claim 42, wherein the female maize parent plant line includesa genetic background of su1su1 se1se1 in its genome.
 50. A method ofclaim 49, wherein the female maize parent plant line includes anendosperm allelic construction of su1su1 se1se1 sh2-i sh2-i.
 51. Amethod of claim 42, wherein the female maize parent plant line includesan endosperm allelic construction of Su1Su1 se1se1 sh2-i sh2-i or su1su1Se1Se1 sh2-i sh2-i.
 52. A method of claim 51, wherein the female maizeparent line includes an endosperm allelic construction of Su1Su1 se1se1sh2-i sh2-i.
 53. A method of claim 42, wherein the male maize parentplant line includes an allelic construction including both of thesugary-1 and sugary enhancer-1 mutant endosperm alleles in homozygouscondition.
 54. A method of claim 42, wherein the male maize parent lineincludes an allelic construction of se1se1 sh2sh2, su1su1 sh2sh2 orsu1su1 Se1Se1 sh2sh2.
 55. A method of claim 54, wherein the male maizeparent plant line includes su1su1 Se1Se1 sh2sh2 in its genome.
 56. Amethod of claim 45 or 46, wherein the male maize parent line includesse1se1 sh2sh2, su1su1 sh2sh2 or su1su1 Se1Se1 sh2sh2 in its genome. 57.A method of claim 49, wherein the male maize parent line includes se1se1sh2sh2, su1su1 sh2sh2 or su1su1 Se1Se1 sh2sh2 in its genome.
 58. Amethod of claim 50, wherein the male maize parent line includes se1se1sh2sh2, su1su1 sh2sh2 or su1su1 Se1Se1 sh2sh2 in its genome.
 59. Amethod of claim 52, wherein the male maize parent line includes se1se1sh2sh2, su1su1 sh2sh2 or su1su1 Se1Se1 sh2sh2 in its genome.
 60. Amethod of claim 42, wherein the male parent line includes an allelicconstruction including both of the sugary-1 and sugary enhancer-1 mutantendosperm alleles in homozygous condition.
 61. A method of claim 56wherein the male maize parent plant line includes su1su1 sh2sh2 orsu1su1 Se1Se1 sh2sh2 in its genome.
 62. A method of claim 42, whereinthe method includes the use of molecular markers to identify a maizeplant that includes any of said mutant endosperm alleles in its genome.63. A method of claim 42, wherein molecular markers are not used.
 64. Amethod of claim 42, wherein the hybrid maize plant, plant material orseed is a sweet corn plant, plant material or seed.
 65. A method ofclaim 56, wherein the hybrid maize plant, plant material or seed is asweet corn plant, plant material or seed.
 66. A method of claim 64,wherein the sweet corn plant, plant material or seed is classified asZea mays var. rugosa.
 67. A method of claim 65, wherein the sweet cornplant, plant material or seed is classified as Zea mays var. rugosa. 68.A method of claim 42, wherein the maize plant, plant material or seedhas an enhanced ability to retain sugar in days 1-7, days 1-14 or days7-14 immediately following a prime eating stage thereof in comparisonwith a conventional mutant sugary-1 or shrunken-2i maize plant, plantmaterial or seed.
 69. A method of claim 42, wherein the maize plant,plant material or seed has an enhanced vigor in comparison with aconventional mutant shrunken-2 maize plant, plant material or seed andone or more other desirable traits.
 70. A maize plant produced by themethod of any one of claims 1-37, 38-41, 44, 42, 46-48, 49, 50-55, 57,58-60, 62-64, 66 and 68-69.
 71. A maize plant part produced by themethod of any one of claims 1-37, 38-41, 44, 42, 46-48, 49, 50-55, 57,58-60, 62-64, 66 and 68-69.
 72. A maize seed produced by the method ofany one of claims 1-37, 38-41, 44, 42, 46-48, 49, 50-55, 57, 58-60,62-64, 66 and 68-69.
 73. A maize plant produced by the method of claim45.
 74. A maize plant part produced by the method of claim
 45. 75. Amaize seed produced by the method of claim
 45. 76. A maize plantproduced by the method of claim
 43. 77. A maize plant part produced bythe method of claim
 43. 78. A maize seed produced by the method of claim43.
 79. A maize plant produced by the method of claim
 61. 80. A maizeplant part produced by the method of claim
 61. 81. A maize seed producedby the method of claim 61.